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Bio 08 Western blot Detection using CCD digital imaging Fast purification of proteins with magnetic beads Comparative performance Evaluation of MSIA™ & Magnetic Bead Formats Offices & Service Centers Australia Head Office: Street Address: 5 Caribbean Dr, Scoresby, Victoria 3179 Postal Address: PO Box 9092, Scoresby, Vic 3179 Telephone Number: 1300-735-292 Service & Calibration:1300-736-767 AU Service Centre Locations: Melbourne: Scoresby Sydney: North Ryde Brisbane: Richlands South Australia: Thebarton Perth: Malaga New Zealand Head Office: Street Address: 244 Bush Road, Albany, North Shore City, 0632, Postal Address: Private Bag 102922, North Shore, North Shore City, 0745 Telephone Number: 0800-933-966 NZ Service Centre Locations: Auckland: Albany Welcome This publication is the eighth edition of the Bio-innovation series, a magazine dedicated to discussing new and emerging trends relevant to the field of life science for our Australian and New Zealand customers. The focus of this edition is advances in technology in Protein Research. We have included articles covering both upstream and downstream processes, as well as sample preparation. There are articles comparing various techniques such as isolating small quantities of protein from complex samples; comparing differences in optical density analysis between spectrophotometric instruments; and presenting different methods of protein quantification. We are excited to introduce the myECL imager, our new chemiluminescent instrument and show its superior performance to X-ray film. There is also an informative piece about our Superspeed Centrifuge and how this equipment can be used to improve large scale recombinant protein production. Finally we discuss the performance of our plasticware for stem cell growth and sample preparation against other plastics. Thermo Fisher Scientific is here to help you by providing the most cutting-edge options available. I hope this edition of Bio-innovation gives you some new insights into ways to approach your research and achieve your goals. Christchurch: Wigram Palmerston North Wellington: Lower Hutt Editor Mika Mitropoulos: [email protected] Art & Design Andrew Dennis: [email protected] Cover Image Grass pollen allergen. Molecular model of the major grass pollen allergen Phl p2 complexed with the antigen-binding fragment (fab) of its human immunoglobulin E antibody. Phl p2 is responsible for allergic reactions to grass pollen. Robyn Matthews Product Manager - Proteomics Thermo Fisher Scientific Contents Western blot detection using CCD digital imaging .................................................................04 Evaluation of the MSIA™ & magnetic bead formats..............................................................08 Large scale recombinant protein production.........................................................................11 Microvolume quantification of proteins by UV-VIS absorbance or fluorescence.........................14 Product focus: Fisherbrand® shaker flasks...........................................................................17 How it works: superspeed centrifuge ..................................................................................18 Protein Biology Workflow....................................................................................................20 Culturing embryonic stem cells ..........................................................................................26 Differences in bacterial optical density measurements between spectrophotometers...............30 Fast purification of proteins with magnetic beads .................................................................34 Benefits of low retention microcentrifuge tubes....................................................................38 Total protein quantitation ...................................................................................................40 Western blot detection using CCD digital imaging western blot detection using CCD digital imaging Comparison of cooled-CCD digital imaging versus X-ray film for sensitivity, dynamic range and signal linearity with enhanced chemiluminescence (ECL). Chemiluminescent Western blotting is a popular protein detection method for studying proteins. The typical protocol for measuring chemiluminescent signal exposes the Western blot to laboratory-grade X-ray film, but digital imaging using a cooledCCD camera is proving to be a more functional choice for researchers. The Thermo Scientific myECL Imager offers digital imaging with a number of improvements over film detection, including greater signal sensitivity, linearity and dynamic range. Western blot analysis has been used as a qualitative assessment of the presence of a specific protein of interest in a sample with a target-specific antibody. Relative differences in protein amounts can be assessed by the signal intensity as it typically correlates to the protein amount in the blot. Scanning of the film with a basic document scanner and the use of image analysis software has been used to extract some quantitative information; however, densitometry from the scanned X-ray film image can be challenging (Ref.1) and chemiluminescent Western blot analysis is generally regarded as not being quantitative. An alternative method to X-ray film for capturing luminescent 4 signals from Western blots is to use a cooled-CCD camera. Charge-coupled device (CCD) cameras use a light-sensitive silicon chip that converts photons into digital signals. Imagers with early generation CCD cameras designed for low-light applications were not capable of matching the speed or sensitivity of X-ray film. Recent improvements in CCD technology have enabled the development and commercialisation of sensitive, cooled-CCD cameras with higher light-capturing performance than film. High-performance CCD cameras cool the silicon chip to sub-zero temperatures to reduce dark current, which produces background noise. To enhance detection sensitivity further, the pixels, or light capturing units of the CCD chip, can be combined or binned. Binning increases the size of each pixel, which effectively increases the amount of light collected in the pixel area. Combining a highly sensitive CCD camera and digital image analysis software, researchers who perform chemiluminescent Eric Hommema, Ph.D.; Nikki Jarrett, M.S.; Steve Shiflett, Ph.D.; Suk Hong, Ph.D.; Priya Rangaraj, Ph.D.; Brian Webb, Ph.D.; June 26, 2013 Western blot analysis can have the ability to extract more accurate qualitative and quantitative information than was previously available with film. In this article, we demonstrate that the myECL Imager is superior to X-ray film in sensitivity, dynamic range and signal linearity. RESULTS and DISCUSSION: To compare CCD camera and X-ray film imaging, we examined signals produced by a NIST-traceable Luminometer Reference Microplate (Harta Instruments, #RM-168) and by our own chemiluminescent Western blots containing serially diluted samples spanning the detectable range for imagers and film. We captured signals digitally using the myECL Imager and by exposure to X-ray film. (Developed films were then scanned at high resolution to produce digitised versions for analysis.) Finally, we analysed the images using the Thermo Scientific myImageAnalysis Software. Greater sensitivity & dynamic range compared to X-ray film Images acquired on the myECL Imager are more sensitive than film. Seven of the eight reference plate spots are visible and distinguishable above background on the 300-second exposure acquired by the myECL Imager (Figure 1). In contrast, only five of the eight spots are visible on a 300-second exposure to film. Densitometry of these images shows a 1000-fold dynamic range (maximal signal intensity/minimal signal intensity) in the myECL Imager compared to a 1.5-fold dynamic range for the film, where dynamic range is calculated from the density of spot 1 divided by the density of spot 8 (Figure 1; Table 1). A qualitative assessment of the Western blot examples shows that the myECL Imager detects an additional band of the HeLa lysate dilution series when compared to film (Figure 2). Figure 1. Western blot detection using CCD digital imaging Figure 1. Image sensitivity and dynamic range comparison of film and the Thermo Scientific myECL Imager. Panel A. At standard binning (3 × 3), images of the Luminometer Reference Microplate on the myECL Imager show greater sensitivity than those from film. Panel B. Image analysis of the Reference Luminometer Plate images demonstrate the greater dynamic range of signal using the myECL Imager compared to film. Table 1. Densitometry values for 300-second exposures of reference plate. Results show a 1000-fold (45017/45) dynamic range for the myECL Imager and a 1.5-fold (62734/41286) dynamic range for X-ray film. 5 Western blot detection using CCD digital imaging Figure 2. Sensitivity comparison between CCD imaging and X-ray film. Western blot images of 2-fold serially diluted HeLa lysate probed with anti-PLK1 (Panel A) and anti-Cyclophilin B (Panel B) are shown. Blots were incubated with SuperSignal West Dura Substrate and exposed for 10 seconds to film and the myECL Imager (3 × 3 binning). Figure 3. Result of binning on imaging sensitivity. The Luminometer Reference Microplate was imaged on the myECL Imager for 30 seconds on each of the five binning settings and on film. More pixel binning results in more sensitivity with a corresponding increase in the pixel size. The myECL and film images were adjusted to optimal contrast for display. Figure 2. High pixel binning in the Figure 3. myECL Imager increases imaging sensitivity Pixel binning is the ability to combine signals from adjacent pixels to increase the light capturing ability (sensitivity) of a CCD chip. Conversely, pixel combination will result in a larger pixel, which then results in a lower resolution of the captured image. Each binning increase results in an increase in light capturing ability or sensitivity (Figure 3). Adjustment of the binning setting allows the researcher to find the desired balance between sensitivity, resolution and exposure time. We found the default 3 × 3 setting generally offers the best combination of sensitivity and resolution when using the myECL Imager. CONCLUSIONS: Life science researchers have traditionally used X-ray film for Western blot analysis. This provides qualitative data for protein detection, but film limits protein quantitation because of narrow dynamic range and low sensitivity. Additional drawbacks of film, such as dealing with hazardous developing solutions, 6 maintaining film processors and needing a dark room, make digital imaging easier and more practical. The myECL Imager, with a broad and linear dynamic range, allows researchers to extract quantitative data from Western blots with the additional benefits of convenience and sensitivity. METHODS: Luminometer Reference Plate Time Course A Luminometer Reference Microplate (Harta Instruments, #RM-168) was used to demonstrate the imaging speed and sensitivity of the myECL Imager in comparison to standard X-ray film (Thermo Scientific CL-XPosure Film, Part No. 34090). The reference plate contains eight lights (spots) of varying, known signal intensities and is typically used for luminometer calibration. Images were captured on film and on the myECL Imager using all five binning settings (1 × 1, 2 × 2, 3 × 3, 4 × 4, and 8 × 8) at exposure times of 10, 30, 60, 120 and 300 seconds. The film was developed in a Konica Minolta™ SRX-101A Film Processor with developer reagents. To create digital images of the film for analysis, each film was scanned with an Epson™ 4990 Photo Scanner as a 300 PPI (pixels/inch), 16-bit grayscale TIFF image to match the image output from the myECL Imager. Image analysis was performed using the Thermo Scientific myImageAnalysis Software. For analysis, a manual region of equal size was placed over each of the eight reference plate light spots and the density (pixel intensity/region area) was plotted against the observed RLU (Relative Light Units) based on the manufacturer’s Certificate of Analysis. For publication, the white level contrast was adjusted on the images acquired with the myECL Imager to display low-intensity pixels. Western Blots Fifty micrograms of HeLa lysate was serially diluted (2-fold in each lane) in 4X LDS Sample Buffer (Product # 84788) supplemented with DTT at a final concentration of 50mM. The amount of sample loaded onto the gel was 50, 25, 12.5, Western blot detection using CCD digital imaging 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977µg of lysate. Samples were loaded onto 4-20% Thermo Scientific Precise Tris-Glycine Gels (Part No. 25249) and transferred to Thermo Scientific PVDF Transfer Membrane (Part No. 88518) using the Thermo Scientific Pierce G2 Fast Blotter (Part No. 62288) and 1-Step Transfer Buffer (Part No. 84731). The blots were probed with mouse anti-PLK1 (Ab No. MA1-848) or rabbit anti-Cyclophilin B (Ab No. PA1-027A) according to instructions supplied with the Thermo Scientific Fast Western, SuperSignal West Dura Kit (Part No. 35070) for 1 hour. The anti-mouse or anti-rabbit fast Western optimised horseradish peroxidase (HRP) reagents were added for 10 minutes, followed by four 5-minute washes with Thermo Scientific Fast Western Wash Buffer. SuperSignal West Dura Substrate was added for 5 minutes before imaging on film, followed by image capture using the myECL Imager. Signal loss between imaging events (approx. 5 minutes) was negligible (data not shown). Images were acquired on the myECL Imager in Chemi mode with the default binning setting (3 × 3). X-ray film was developed and scanned as described above. Signal Linearity A serial dilution of 2.0 to 0.004ng of in-vitro-expressed GFP was loaded onto 4-20% Precise Tris-Glycine Gels and transferred to nitrocellulose membrane using the Pierce G2 Fast Blotter and 1-Step Transfer Buffer. The blot was probed with Thermo Scientific Mouse anti-HA Antibody (Part No. 26183) according to instructions supplied with the Fast Western, SuperSignal West Dura Blot Kit for 1 hour. The anti-mouse fast Western optimised HRP reagent was added for 10 minutes, followed by four 5-minute washes with Fast Western Wash Buffer. SuperSignal West Dura Substrate was added for 5 minutes before imaging on film, followed by the myECL Imager. Signal loss between imaging events (approx. 5 minutes) was negligible (data not shown). For analysis, a manual region of equal size was used over each of the GFP-HA bands in all 10 lanes plus a representative background region. The Global Background Subtracted Density (pixel intensity/region area) was plotted against nanograms of GFP-HA. For publication, the white-level contrast was adjusted on the images acquired with the myECL Imager to display low-intensity pixels. in-vitro Protein Expression The Thermo Scientific 1-Step Human in-vitro Protein Expression Kit (Part No. 88882) was used to express green fluorescent protein (GFP) with a C-terminal HA tag. XhoI and NdeI restriction sites were added to the 5’ and 3’ ends, respectively, to the open reading frame of GFP from Pontellina plumata. This fragment was inserted into the multiple cloning site of the pT7CFE-CHA expression vector, which contains the upstream transcription and expression elements necessary for in-vitro translation and a C-terminal HA (Human Influenza Hemagglutinin-YPYDVPDYA) tag. Protein expression was performed as described in the kit protocol. After protein expression, the concentration of GFP was determined by measuring fluorescence (482ex/502em) in a Microplate Reader and compared to a GFP standard. For more information contact [email protected] CITED REFERENCES: 1.Gassmann, M., Granacher, B., Rohde, B. and Vogel, J. (2009) Quantifying Western blots: Pitfalls of densitometry. Electrophoresis 30:1845-55. Editor’s Note:This article was first printed as Application Note 1602635, April 2013. 7 Evaluation of the MSIA™ & magnetic bead formats V Comparative performance Evaluation of the Mass Spectrometric Immunoassay (MSIA™) & Magnetic Bead Formats The use of Mass Spectrometry (MS) in protein/ peptide detection has become the staple in proteomics applications. The analysis of selected proteins from complex biological fluids is readily achievable using LC/MS/MS methods. These methods have repeatedly demonstrated the ability to detect thousands of proteins/ peptides from a single sample; however, the complexity of these samples has made routine & consistent analysis troublesome. In order to overcome these issues, the introduction of immunoaffinity capture and enrichment as a part of the front-end processing has become widely adopted. Now known as the Mass Spectrometric Immunoassay (MSIA)1-4, this specific target enrichment procedure provides a cleaner and more consistent sample for analysis, and in addition, allows for the detection of much lower abundant proteins not previously 8 achievable in proteomics applications. It is, therefore, advantageous to have a highly efficient, reproducible, and effective technology to perform such an enrichment step. Presented here is a description of a fast and effective approach to performing immunoaffinity sample purification. Using a novel immunoaffinity enrichment technology called the Thermo Scientific™ Protein A/G MSIA™ Tips, a head-to-head comparison against traditional Protein A/G magnetic beads was performed. In this study, we specifically examined the lower limits of detection and quantification (LLOD and LLOQ, respectively), as well as non-specific binding of both analyte extraction formats using an Insulin-Like Growth Factor 1 (IGF1)5-6 model system. This testing demonstrated that the MSIA-Tips provide a 10-fold improvement in the LLOD and a 20-fold improvement in the LLOQ over a comparable volume of beads. The MSIA-Tips also provided higher quality immuno-affinity purification that resulted in less non-specific binding. This resulted in a 55% increase in IGF1 selectivity over the beads tested. Evaluation of the MSIA™ & magnetic bead formats S Materials • Thermo Scientific Protein A/G MSIA-Tips • Thermo Scientific Versette Liquid Handling Platform • BcMag™ Protein A/G Magnetic Beads (Bioclone Inc.) • Magnetic Stand • Anti-human IGF1 antibody • Human recombinant IGF1 (IGF1 standard) • Recombinant LR3-IGF1 (Internal reference standard) • Human EDTA Plasma • Thermo Scientific TSQ Vantage™ Triple Stage Quadrupole Mass Spectrometer • Thermo Scientific LTQ Orbitrap™ XL • Thermo Scientific Hypersil GOLD™ C18 column (50 mm x 2.1 mm, 1.9 μm particle size) Method Antibody Loading The BcMag™ Protein A/G Magnetic Beads and the Protein A/G MSIA-Tips utilised the same affinity reagents in this study. Both were loaded with anti-IGF1 antibody using the recommended manufacture’s protocol. In order to provide more consistent analyses, a magnetic stand was used in the bead applications while the Versette platform was used with the tips. Extraction and Enrichment Once loaded, both formats were ready for sample interrogation. However, in order to perform a representative head-to-head comparison, the volume of beads used was scaled in order to match the fixed volume of the micro-columns within the MSIA-Tips prior to application. The bead volume required was 0.6 µL, which was calculated based on the published binding capacity values for both technologies. The samples used, calibrants (1.0, 2.5, 5.0, 10 and 20 µg/L) and plasma samples, were prepared following the protocol provided in Technical Note: MSIA1001. In these experiments, the calibrants were analysed in triplicate (n = 3) and used for the determination of both the LLOD and the LLOQ. The human plasmas were used for the background assessment and were performed in decaplet (n = 10). The beads were applied following the manufacturer’s suggested protocol and the Protein A/G MSIA-Tips were used as described in Technical Note: MSIA1001. Post incubation, both formats were subject to a series of rinse steps followed by elution. The same rinse and elution buffers were used with each. The extracted proteins using both methods were then subjected to identical post extraction processing (i.e., reduction, alkylation, trypsinisation, etc.). These processing protocols can also be found in Technical Note: MSIA1001. Urban A. Kiernan, Senior Scientist, David A. Phillips, Technician, Dobrin Nedelkov, Manager of Research and Development and Eric E. Niederkofler, Senior Scientist, Thermo Fisher Scientific, Tempe, Arizona. Bryan Krastins, Senior Applications Scientist, David A. Sarracino, Senior Applications Scientist, Scott Peterman, Senior Applications Scientist, Amol Prakash, Senior Applications Scientist, Alejandra Garces, Senior Applications Scientist, and Mary F. Lopez, BRIMS Center Director, BRIMS Center, Thermo Fisher Scientific, Cambridge, Massachusetts. Analysis In this study, two forms of LC-MS/MS analysers were used: 1) Thermo Scientific LTQ Orbitrap XL – for the assessment of format non-specific binding, 2) Thermo Scientific TSQ Vantage Triple Stage Quadrupole Mass Spectrometer – used for SRM analysis in the determination of the LLOD and LLOQ of each affinity format. Both MS/MS systems utilised a Thermo Scientific Accela™ pump, a CTC PAL® autosampler, and a Thermo Scientific Hypersil GOLD C18AQ fused silica capillary column for the LC systems. LC-MS/MS runs were performed using standard protocols for the analysis of the IGF1 tryptic peptides of interest (originating from IRS and human), as described in Technical Note: MSIA1001. 9 Evaluation of the MSIA™ & magnetic bead formats Figure 2. Representative data generated using the TSQ Vantage of the same IGF1 peptide. The tip extraction method resulted in more uniform peak shape and less interference within the elution window than with the beads. Table A. The established LLOD and LLOQ of both the Protein A/G MSIA-Tips and the BcMag™ Magnetic Bead immunoaffinity extraction systems. References 1.Nelson, R.W.; Krone, J.R.; Bieber, A.L.; Williams, P. Mass spectrometric immunoassay Anal. Chem. 1995, 67, 1153– 1158. 2.Niederkofler, E.E.; Tubbs, K.A.; Gruber, K.; et al. Determination of x−2 Microglobulin Levels in Plasma Using a High-Throughput Mass Spectrometric Immunoassay System. Anal. Chem. 2001, 73, 3294-3299. 3.Niederkofler, E.E.; Kiernan, U.A.; O’Rear, J.; et al. Detection of Endogenous B-Type Natriuretic Peptide at 1.Very Low Concentrations in Patients With Heart Failure. Cir. Heart Fail. 2008, 4, 258-264. 4.Lopez, M.F.; Rezai, T.; Sarracino, D.A.; et al. Selected Reaction Monitoring–Mass Spectrometric Immunoassay Responsive to Parathyroid Hormone and Related Variants. Clin. Chem. 2010, 56, 281-290. 5.Monzavi, R.; Cohen, P. IGFs and IGFBPs: role in health and disease. Best Pract. Res. Clin. Endocrinol. Metab. 2002, 16, 433-447. 6.Hankinson, S. E.; Willett, W. C.; Colditz, G. A., et al. Circulating concentrations of insulin-like growth factor-1 and risk of breast cancer. Lancet. 1998, 351, 1393-1396. 10 Results and Discussion Assessment of Non-Specific Binding Data for the assessment of non-specific background interference was obtained using both LC-MS/MS systems. Figure A shows representative Mass Spectra observed from the Protein A/G MSIA-Tips and the Protein A/G BcMag Magnetic Beads extracted samples using the LTQ Orbitrap XL. The MSIA-Tips were able to more efficiently extract the IGF1 from the samples when compared to the beads. As shown in (Figure 1: Top), there was far less background using the tips than observed with the bead method (Figure 1: Bottom). The data presented showed a 55% improvement in the IGF1 signal vs. the detected background. This decrease in non-specific binding provides a clear analytical benefit as the improved percent selectivity will translate into improved detection and signal stability in SRM analyses. Figure 1. Figure 2. This sensitivity was assessed using SRM analysis, in which the LLOD and the LLOQ of both assay extraction formats were determined. These characteristics were determined by evaluating the standard deviations of the sample replicates at each of the calibration points. An observed overlap of ≥ 50% between successive calibration points was used as a threshold for establishing both points. Using these metrics, data clearly showed a ≥ 10-fold improvement in both the LLOD and the LLOQ using the Tips vs. the Bead format. This data is presented in Table A. Table A Figure 1. Resultant LTQ Orbitrap XL MS/MS data of IGF1 obtained from both the MSIA-Tips (Top panel) and BcMag™ Magnetic Bead (Bottom panel) protein capture and enrichment. Data clearly shows less interference for the Tip extraction vs. the Bead when detecting the peptide of interest. When the SRM analysis was performed on the same peptides using the TSQ Vantage, the data supported the previously discussed benefits. This is shown in Figure 2, in which the representative chromatograms of the SRM analyses illustrate the influence of these non-specifically retained contaminants. The observed chromatograms from the bead extractions showed large inconsistencies between the two extraction formats within the MS window of the IGF1 target peptide. Moreover, the problem is compounded by the presence of additional contaminant peaks, as both issues limited the sensitivity and the accuracy of the assay. Extraction Format Manufacturer LLOD (ng/mL) (n = 3) LLOQ (ng/mL) (n = 3) MSIA-Tips Thermo Fisher Scientific 1 1 BcMag™ Magnetic Beads Bioclone Inc. 10 20 Conclusion The Protein A/G MSIA-Tips demonstrated the ability to generate enhanced results over the BioClone Protein A/G BcMag™ Beads. Not only were the MSIA-Tips able to demonstrate superior LLOD and LLOQ using a standard tryptic digest LC-MS/MS SRM workflow over a comparable capacity of beads, but more specific signal due to decreased nonspecific binding. Both these benefits were directly associated to one another, and these results clearly demonstrated such technical benefits which instill confidence in the data generated by the end user. Other characteristics of this model system using the Protein A/G MSIA-Tips were established and can be found in Technical Note: MSIA1002. Large scale recombinant protein production Large Scale Recombinant Protein Production Large scale recombinant protein production is becoming increasingly important for applications in the field of proteomics. Protein characterisation and functional studies can provide clues about the mode of action and can facilitate development of therapeutic drugs. Large scale recombinant protein production is becoming increasingly important for applications in the field of proteomics. Protein characterisation and functional studies can provide clues about the mode of action and can facilitate development of therapeutic drugs. For instance, using structural biology, inhibitors can be made that bind to, and inactivate, disease-related enzymes. Additionally, analysis of protein-DNA interactions enables scientists to better understand the expression of disease-related genes. In other cases, protein structure has aided in the study of antigenantibody interaction and ligand-receptor binding, helping to understand immunology and molecular signal transduction. However, to successfully study protein structure and function, large quantities of protein (mg to g amounts) must be isolated from an appropriate expression system, such as a bacterial (e.g. Escherichia coli) or eukaryotic cell-based expression system (e.g. yeast, insect, mammalian). In order to meet this need, cell cultures can be grown in large volume in a shake flask culture or by the implementation of fermentation techniques. Using high cell density or fermentation, a researcher can optimise cell culture conditions to increase cell mass and recombinant protein yield. The following protocols describe the usage of the Thermo Scientific Sorvall LYNX 6000 superspeed centrifuge and large volume Thermo Scientific Fiberlite rotors to increase the efficiency of cell harvesting prior to protein isolation. MATERIALS AND METHODS During high volume cell culture, translated recombinant protein is secreted into the culture medium and/or remains intracellular. In either case, large scale centrifugation marks the first step in preparing the recombinant protein. Listed below are basic protocols for protein production in large volume bacterial cell cultures (up to 6 L). PROCEDURES Protocol 1: Basic Protocol for the Production of Recombinant Protein in Bacterial Cells. Protocol Adapted from Current Protocols in Protein Science, Bernard and Payton, 1995-1 1. Inoculate 100 mL flask containing 20 mL LB medium with Escherichia coli (E. coli) expressing the gene of interest. Shake overnight at 37°C. Note: The protocol for the transformation of E. coli with a gene of interest is detailed in protocol 3. Other methods for the induction of gene expression in bacterial cells can be conducted using many commercially available transformation kits. 2. Transfer overnight culture to 800mL LB medium in a 3L Erlenmeyer flask. Incubate and shake cells 1 to 2 hr at 37°C. 3.After cells reach exponential growth phase (OD600 of 0.5 to 0.6), transfer cells to a 500mL – 1L chilled centrifuge bottle. 4.Place centrifuge bottles in the Thermo Scientific Fiberlite F9-6x1000 LEX, F10-4x1000 LEX, or F12-6x500 LEX rotor and perform centrifugation with the Sorvall® LYNX 6000 superspeed centrifuge with the following parameters: 4000 x g for 15 min at 4°C. 5.Resuspend the pellet with the appropriate medium for subsequent protein analysis. 11 Large scale recombinant protein production Figure 1. 4-20% SDS-PAGE of IL-8 NiNTA Purification. 20 mg of IL-8 was produced in a 4 L fermentation run, which is a tenfold increase in production over a previous shake flask culture of the same total volume. parameters alone will increase cell density at least six-fold (unpublished observations), and we expect a concomitant increase in recombinant protein yield. Figure 1. 1. Bio-Rad® Prestained MW Marker 6. NiNTA Flow Through 3 2. IL8 Standard (2 μg) 7. NiNTA Wash 1 3. NiNTA Load 8. NiNTA Wash 2 4. NiNTA Flow Through 9. NiNTA Eluate 1 5. NiNTA Flow Through 2 10. NiNTA Eluate 2 Protocol 2: Utilisation of Fermentation to Enhance Recombinant Protein Production Protocol Adapted from Current Protocols in Protein Science, Bernard and Payton, 1995-2 Kelli Benge, Research Associate, Zyomyx, Inc., Hayward, CA 94545 In a recent recombinant human Interleukin-8 production run, our group achieved a two-fold increase in the cell density of a 4 L batch culture as compared to a previous shake flask experiment, and a ten-fold increase in rhIL-8 yield (Figure 1). The fermentation timeline followed that of the shake flask experiment, and no nutrient additions were made to the media. The only differences in the experiments were that the dissolved oxygen was controlled at 30% (vs. uncontrolled in the shake flask), and the temperature was maintained at 32°C instead of 37°C during the cell growth phase. To optimise culture conditions in future experiments, pH will be regulated and glucose will be added to the media. Optimising these two 12 A basic protocol for utilisation of fermentation to produce results similar to those above is as follows (please follow manufacturer’s instructions for fermentation use): 1. Prepare the inocolum and fermentor. a. Prepare an overnight culture of transformed E. coli strain expressing the protein of interest. b. Grow culture until stationary phase is reached (OD600 >1.5). Use overnight culture to inoculate the fermentor (volume is 1-10% of intended fermentation volume). 2.Sterilise the fermentor and medium. 3.Inoculate the fermentor, grow cells, and induce protein expression. a. Inoculate fermentor by transferring inoculum aseptically into reactor vessel. b. Monitor growth by measuring OD600 of samples at 1hr intervals. When target cell concentration is reached, proceed to induction. Target cell concentration may be within an OD600 of 5 to 20. c. Induce protein expression via temperature or chemical induction. 4. Harvest the cells. a. For small fermentors (1-10L) i. Pressurise the fermentor and transfer the reactor contents into an intermediate container. ii. Aliquot culture into 500 mL or 1 L centrifuge bottles. iii.Perform centrifugation using the Sorvall LYNX 6000 superspeed centrifuge and the Fiberlite® F9-6x1000 LEX, F10-4x1000 LEX, or F12-6x500 LEX rotor with the following parameters: 5000 x g for 15 min at 4°C. Large scale recombinant protein production 5. Store the harvested cells and clean the fermentor. a. Collect pellets from each centrifuge bottle and place together in a heat-sealed plastic bag. Pellets may be frozen at -20°C and used for subsequent protein analysis. Protocol 3: Production of Recombinant Protein by Electroporation of E.coli cells Adapted from Current Protocols in Protein Science, Bernard and Payton, 1995-1 1. Prepare competent cells. a. Inoculate 100 mL flask containing 20 mL LB medium with a single colony of the E. coli host strain to be transformed. Shake overnight at 37°C. b. Transfer overnight culture to 800 mL LB medium in a 3L Erlenmeyer flask. Incubate and shake cells 1 to 2 hr at 37°C. c. After cells reach exponential growth phase (OD600 of 0.5 to 0.6), transfer cells to a 500 mL to 1L chilled centrifuge bottle. d. Perform centrifugation with the Sorvall LYNX 6000 superspeed centrifuge and the Fiberlite F9-6x1000 LEX, F10-4x1000 LEX, or F12-6x500 LEX rotor with the following parameters: 4000 x g for 15 min at 4°C. e. Discard supernatant and resuspend pellet in 5-10mL of ice cold water. Bring to a final volume of 800mL with ice-cold water and perform centrifugation with the following parameters: 4000 x g for 15 min at 4°C. Repeat twice. f. Resuspend pellet in ice-cold water to a final volume of 50mL and perform centrifugation in chilled 50mL centrifuge or conical tubes with the Sorvall LYNX 6000 superspeed centrifuge and the Fiberlite F20-12x50 LEX or F14-14x50cy rotor with the following parameters: 4000 x g for 15 min at 4ºC. g. Discard supernatant, estimate the volume of the pellet, and add an equal volume of ice-cold water. h. Vortex, distribute 100 µL aliquots into prechilled 1.5mL sterile microcentrifuge tubes. Cell aliquots may be used immediately or stored with glycerol at -80°C. References1. Bernard, A. and M. Payton (1995-1). Selection of Escherichia coli Expression Systems. Current Protocols in Protein Science 5.2.1-5.2.18. 2. Transform competent cells. a. Add 5 pg to 0.5 µg plasmid DNA to 100 µL of competent cells. Mix by inverting tubes several times. b. Set electropotation apparatus to 2.5 kV, 25 µF, 200 Ω. c. Transfer DNA and cells to prechilled electroporation cuvette, place cuvette into electroporation chamber and apply the electrical pulse. d. Remove cuvette, add 1 mL SOC medium and transfer contents to a 20 mL sterile culture tube. Incubate 60 min with moderate shaking. e. Plate transformation culture (10 to 100 µL) onto LB plates to isolate single colonies. Single colonies can then be picked to produce pure and viable cultures of the bacteria expressing your protein of interest. 2. Bernard, A. and M. Payton (1995-2). Fermentation and Growth of Escherichia coli for Optimal Protein Production. Current Protocols in Protein Science 5.3.1-5.3.18. Conclusion In order to produce large quantities of protein for proteomics work, cell cultures must be large volume or cell dense. Fermentation improves efficiency by increasing cell density and provides easier handling of large cultures (>4L), since the culture is contained in one vessel as compared to handling several shake flasks. Pelleting of bacterial cells from a large volume culture can be completed in the Fiberlite F9-6x1000 LEX, F10-4x1000 LEX, or F12-6x500 LEX rotors in the Sorvall LYNX 6000 superspeed centrifuge. The Fiberlite carbon fibre rotors are lightweight, and thus, easy to handle, and can process 3-6 L of cell culture in a single run. The time saved in centrifugation with these large volume rotors improves the efficiency of downstream processing of recombinant proteins. 13 Microvolume quantification of proteins by UV-VIS absorbance or fluorescence microvolume quantification of proteins by UV-Vis absorbance or fluorescence Although quantification of proteins using spectrophotometry or fluorometry is commonplace, the choice of technique must be made with several factors in mind. Direct UV A280 absorbance, colorimetric assays, and fluorometric assays are in many cases not interchangeable, and consideration of protein concentration, buffer used, time constraints and sample requirements is necessary. All three types of quantification benefit from the use of NanoDrop™ microvolume capabilities. Microvolume Measurements NanoDrop UV-Vis spectrophotometers utilise a revolutionary sample retention technology which retains 1 – 2 μL samples in place via surface tension between two fiber optic cables. After measurement, samples are quickly and easily removed from the optical surfaces with an ordinary dry laboratory wipe. The final protein concentration, purity ratio, and sample spectra are displayed on user friendly software (fig. 1). Figure 1: Upper left: loading of a 2 μL protein sample on the measurement pedestal, Lower left: liquid column between pedestals during sample measurement, Right: software output, showing both spectra and numerical data Figure 1. The NanoDrop 2000/2000c spectrophotometer utilises multiple pathlengths (1.0, 0.2, 0.1 and 0.05 mm) that change in real time while measuring a 2 μL protein sample (fig. 1), resulting in a wide dynamic range capable of measuring 0.1 – 400 mg/mL of purified BSA protein using the direct UV A280 software module. This automatic pathlength optimisation ultimately eliminates the need for sample dilutions, resulting 14 in greater accuracy. In contrast to this, measuring samples with a standard 10 mm quartz cuvette on a conventional spectrophotometer typically has an upper detection limit of ~1.8 mg/mL and requires 500 fold more sample to meet the minimum sample volume of 1 mL. Moreover, the use of cuvettes can potentially lead to cross-contamination from prior samples if not properly cleaned.The measurement time for the NanoDrop 2000c is less than five seconds, and in cases where higher throughput is desired, the NanoDrop 8000 can measure up to eight samples at a time with a measurement time of just 20 seconds. Moreover, if greater sensitivity is required, microvolume fluorescent protein assays can be accommodated by the NanoDrop 3300 Fluorospectrometer which also measures samples as small as 2 μL. Dynamic Range The typical upper absorbance limit for a spectrophotometer is approximately 1.5 A, which in turn defines the maximum measurable concentration of protein. Measuring protein samples using a fixed pathlength, typically a 1 cm quartz cuvette, limits the linear range of conventional spectrophotometers, resulting in the need for sample dilutions. Such dilutions consume both time and sample, and promote pipetting errors. Measuring purified protein samples with the microvolume sample retention technology used by NanoDrop spectrophotometers by the direct UV A280 method enables the user to rapidly measure up to 400 mg/mL (BSA protein) directly in a 2 μL volume (fig. 2). The upper detection limit of the NanoDrop sample retention technology is 200 times greater than that of a standard 1 cm pathlength quartz cuvette. Microvolume colorimetric assays have shown to be comparable in performance to the same assay performed on a conventional cuvette based spectrophotometer, because even Microvolume quantification of proteins by UV-VIS absorbance or fluorescence David L. Ash and Andrew F. Page | Thermo Scientific NanoDrop Products, Wilmington, DE, USA TABLE 1. Direct UV A280 Absorbance Colorimetric Fluorescence Sample Purity Samples must be purified as contaminants may interfere with measurement. No Purification necessary. No purification necessary. Buffer Compatibility Buffers with strong UV absorbance may be unsuitable (see next panel). Some assays are sensitive to detergents or reducing reagents, which can artificially perturb or enhance colour development. Some assays are sensitive to buffers with primary amines or detergents, which perturb fluorescent signal. Other Considerations Knowledge of an E% value or molecular weight and molar extinction coefficient are required to calculate mg/mL concentration Colorimetric signals vary between proteins, therefore, standards must be carefully chosen in order to minimise differences in signal between the standard and sample proteins. Fluorescent assays typically have a lower detection limit than colorimetric assays or direct UV A280 measurement, but are limited by the maximum measureable protein concentration (fig 2) though the absorbance signal is reduced by approximately 10 fold, the limitation of the assay is normally the assay itself and not the spectrophotometer. Colorimetric assays are typically used to negate the presence of contaminants in unpurified protein samples, however these assays have a limited dynamic range when compared to direct UV A280 measurement (fig. 2). Similarly, the NanoDrop 3300 Fluorospectrometer has also shown comparable performance versus cuvette based fluorometers for most fluorescent protein assays. Fluorescent quantification assays are also used for unpurified protein samples & have shown to have greater sensitivity than both direct UV A280 & colorimetric methods (fig. 2). Figure 2. Sample Requirements The choice of quantification method is heavily influenced by sample requirements (table 1). Although colorimetric Table 1: Sample requirements for direct UV A280 absorbance, colorimetric and fluorescence methods broken down into sample purity, buffer compatibility and other considerations and fluorescent assays may be performed on unpurified protein solutions, direct UV A280 absorbance is only suitable for purified protein solutions. Buffer choice and other considerations, such as sample concentration, are also important. It is always advisable to consult the assay manufacturer for the specific tolerances for buffer components and contaminants for the colorimetric and fluorescent assays. Buffer Compatibility Example Introduction: Commonly used protein buffers, such as RIPA, produce strong absorbance signals in the UV wavelength range (fig. 3A), therefore negatively influencing the accuracy of direct UV A280 measurements. Accurate quantification is possible, however, by utilising a compatible colorimetric assay. This experiment uses both A280 and the BCA colorimetric assay to quantify two protein samples with the same concentration; one in PBS and the other in RIPA buffer. Materials & Methods: A single 2 mg/mL BSA protein standard (Thermo Scientific Pierce Products) was diluted 1:1 with either PBS or RIPA buffer. Standard curves were then created by preparing serial dilutions using the appropriate buffer diluted to 0.5x. Two BSA samples with the same concentration were prepared, one in 0.5x PBS and the other in 0.5x RIPA buffer. Both samples were then quantified by direct UV A280 measurement and also using a BCA colorimetric assay with the relevant standard curve. Figure 2: Comparison of the approximate dynamic ranges associated with the various protein quantification methods using a NanoDrop spectrophotometer or spectrofluorometer 15 Microvolume quantification of proteins by UV-VIS absorbance or fluorescence Table 2: Time requirements for direct UV A280 absorbance, colorimetric and fluorescence methods broken down into preparation, incubation and measurement times. Figure 3: Influence of buffers on direct UV A280 protein measurements, A) Absorbance of 0.5x RIPA buffer (instrument blanked using water), B) Absorbance of the same protein sample in either 0.5x PBS or 0.5x RIPA buffer (blank and sample measurement performed using the same buffer). Note that even when using the appropriate blank, the absorbances of the two samples are not the same. Figure 4: Quantification of the same protein sample in either 0.5x PBS or 0.5x RIPA buffer. Quantification was performed using both A280 (blank performed using the appropriate buffer) and the BCA colorimetric assay (standard curve prepared using the same buffer). Note that the A280 measurement of the sample in RIPA buffer was not only inaccurate, but also showed poor reproducibility. n = 3 for all; error bars represent standard deviations Figure 5: Colorimetric assay development time for Bradford and Pierce 660 assays. Note that both time for colour development and stability of colour vary between assays. TABLE 2. Direct UV A280 Absorbance Colorimetric Fluorescence Preparation None Production of working solutions is required in some assays; fresh standards need to be made for a calibration curve. Production of working solutions is required in some assays; fresh standards need to be made for a calibration curve. Incubation None Time required to stabilise colorimetric signal is between 10 and 60 minutes, depending on the assay (fig. 5) Time required to stabilise fluorescent signal is 10 to 30 minutes, depending on the assay. Measurement ~ 5 seconds using a NanoDrop Colorimetric assays require more time than the direct UV A280 method as a calibration curve must be established prior to unknown sample quantification. Fluorescent assays require more time than the direct UV A280 method as a calibration curve must be established prior to unknown sample quantification. Results: When the protein samples were measured after performing a blank measurement with the appropriate 0.5x buffer, a deviation in sample signal was observed across the monitored wavelength range (fig. 3B). Moreover, the percent difference in concentration derived from the direct UV A280 measurements of the two BSA samples was more than 20%. In addition, precision of sample replication was also compromised for the sample in RIPA buffer (fig. 4). Conversely, quantification of the two protein samples using the BCA colorimetric assay showed the unknown sample to have the same concentration, regardless of buffer (fig. 4). Figure 5. Buffer Compatibility Conclusion: The discrepancy in concentration measurements of the two protein samples when measured by direct UV A280 absorbance measurement is most likely due to the NP-40 or Triton X-100 content of the RIPA buffer, as surfactants such as these strongly absorb UV light. Choice of quantification method is crucial when working with these surfactants, as blanking on any spectrophotometer when using the direct UV A280 method may not fully compensate for the absorbance of the buffer. Figure 3. Figure 4. 16 Time to Result The time required to complete an assay is influenced by three major steps post sample extraction: preparation, incubation, and measurement cycle. The direct UV A280 absorbance method is by far the fastest, as the time required to obtain a result is solely based on the time required to complete the measurement cycle. Table 2 compares the time required to perform the three different assay types, showing how aspects of each assay contribute to the overall time needed to complete the assay. Conclusions As molecular techniques used to interrogate proteins evolve to require smaller amounts of starting material, the need for microvolume quantification measurements must follow in parallel. Several options exist for both absorbance and fluorescent measurements to determine the concentration of protein samples post-extraction. Care should be taken, however, in selecting quantification method. Important considerations include sensitivity requirements, buffers used, time constraints and sample purity. In addition to this, a NanoDrop spectrophotometer or fluorometer can be used to further speed measurement, increase measurable concentration range, and save money on consumables and reagents. Consequently, with the advent of the sample retention technology of the NanoDrop product line it is now possible to perform scaled down protein quantification measurements efficiently with a high degree of accuracy. Eliminating sample dilutions necessary to measure protein samples on conventional spectrophotometers plays a major role in determining protein concentrations with minimal error. product focus Fisherbrand® shaker flasks – Reduce the risk of cross contamination The Fisherbrand Shaker Flask is ideal for shaker and suspension cell culture, media preparation and storage. •Certified sterile at SAL 10-6, USP Class VI •Non-pyrogenic and non-cytotoxic •Autoclavable •Molded-in graduations •Made of crystal clear, durable polycarbonate (PC) For more information contact [email protected] Shaker Flask options available from 125mL to 2800mL with plain or baffled bottom with a vented cap. Options also available from 125mL to 1000mL with a plain bottom and non-vented cap. 17 How it works: superspeed centrifuge How it works... Superspeed Centrifuge Problem: in today’s laboratories, safe, efficient sample processing is essential to getting research answers faster. The centrifuge is a staple of these laboratories and critical to this sample processing. Often a shared resource in busy research facilities, the lab centrifuge can be a revolving door of multiple users with varying levels of experience and a range of applications, all requiring a variety of rotors. Yet, the centrifuge is technically complex and can be the source of lab mishaps if used improperly. These everyday challenges can keep lab managers up at night: are all researchers trained on centrifuge use? Are they using the right rotors for their applications and is the centrifuge programmed with the correct application parameters? Are they ensuring the rotors are properly and safely secured in the centrifuge chamber to avoid any potential rotor accidents? 18 Solution: Designed to overcome these obstacles, pushbutton rotor exchange and instant rotor identification are technology innovations featured in Thermo Scientific™ Sorvall™ LYNX superspeed centrifuges. These technologies are designed to simplify centrifuge operation, safeguard daily sample processing and shorten run set-up time— all without impacting the performance required for these research applications. New quick rotor exchange technology, known as Thermo Scientific™ Auto-Lock™, allows researchers to install or remove How it works: superspeed centrifuge Above: A comparison of traditional high speed centrifuge setup and the Thermo Scientific Sorvall Lynx. centrifuge rotors in only three seconds. Traditional rotors are secured using a tie-down system, which bolts the rotor down onto the centrifuge motor shaft. This traditional method requires proper technique, considerable hand strength and significant time, a process which is then repeated several times a day when rotor changes are needed for a different user, application or protocol. Now using titanium latches with Auto-Lock rotor exchange, the rotor is automatically captured and securely locked into the centrifuge without any user manipulation or procedure, improving safety and confidence that the rotor will not loosen during a run. At the end of the run, a simple pushbutton on the rotor releases the locking mechanism, allowing the rotor to be easily and quickly removed from the centrifuge. Another innovation in these superspeed centrifuges is instant automatic rotor identification with Thermo Scientific™ AutoID™. Using reliable, permanent magnets installed on rotors designed for this centrifuge, this technology instantly detects the magnetic pattern as soon as it is secured in the centrifuge and then automatically loads rotor name and specifications into centrifuge parameters. Instant rotor identification saves considerable run set-up time by eliminating the need to search for and input arcane rotor codes. Additionally, traditional centrifuges perform tests during the centrifuge run to confirm the identity of the rotor is aligned with the rotor which has been programmed. This rotor checking process is done by measuring wind resistance or indirectly calculating rotor mass while the rotor is spinning at up to several thousand rpm and can occur a minute or more after the run has started. This means the user has often left the centrifuge and is not present if a problem is detected. Auto-ID also eliminates the potential to over-speed a rotor by making it impossible to accidently enter an incorrect rotor code or rotor speed, common user errors that on traditional systems can prematurely stop a centrifuge run, with consequences ranging from time lost due to an incomplete separation to damage to valuable samples. Auto-Lock rotor exchange and Auto-ID instant rotor identification are examples of technology innovations that simplify centrifuge operation without sacrificing performance—and give lab managers the assurance of proper usage and safety compliance, while simultaneously providing centrifuge users with higher productivity with ease-of-use, and confidence that successful sample processing has taken place. 19 Start your discovery here Performance Simplified MyECL The Thermo Scientific myECL Imager revolutionises image acquisition of protein and nucleic acid blots and gels detected via chemiluminescent, colorimetric or UV light-activated fluorescent substrates or stains. The compact benchtop instrument uses UV and visible light transillumination, specialised filters and advanced CCD camera technology to capture images with high sensitivity and dynamic range. The large, 10.4-inch touchscreen and on-board computer provide an elegant user-interface to program acquisition settings and manage (store and share) image files. Also included with the instrument is a five-computer license for the Thermo Scientific myImageAnalysis Software, a complete and powerful analysis tool. TSU Ultra Low Temperature Freezer Lynx Centrifuge Thermo Scientific TSU Series freezers deliver ultimate protection and This superspeed centrifuge offers exceptional optimum capacity for your most critical samples. The TSU Series performance to meet the evolving application achieve outstanding thermal performance, safety and security needs of academic and research facilities. With its through state-of-the-art engineering. Packed full of features, the versatile, high-throughput sample processing, built- TSU Series has new Cabinet + Vacuum Insulation Panel Technology in safety, and efficiency, and proven reliability, the which increases the internal capacity of 2 inch vials over previous Sorvall LYNX sets the standard for performance. Its generation freezers—gaining up to 76% more capacity in the 100,605 x g performance and maximised capacity same footprint. The innovative, touch-screen user interface allows up to 6 Liters for bottles, tubes or microplates. Plus monitoring of the freezer’s health 24/7 and access a detailed event breakthrough rotor innovations including Auto-Lock log—simply touch the heart icon on the main screen to see freezer’s rotor exchange, Auto-ID instant rotor identification temperature, access settings and operating parameters. Additional and lightweight and durable Thermo Scientific security options can be added with swip card user access to enable Fiberlite carbon fibre rotors that shorten run set-up full users monitoring. time and increase rotor security. NanoDrop Lite The Thermo Scientific NanoDrop Lite Spectrophotometer is a compact, personal UV-Vis microvolume spectrophotometer that complements the full-featured NanoDrop 2000/2000c and NanoDrop 8000 instruments. The NanoDrop Lite provides rapid, accurate and reproducible microvolume measurements without the need for dilutions. It uses the same sample retention system that has become a hallmark of NanoDrop instruments; the sample is held in place by surface tension only.The NanoDrop Lite performs basic microvolume measurements. Its compact design, with built-in controls and software, make the NanoDrop Lite small enought to fit on any benchtop. The patented sample retention system allow sample to be pipetted directly onto the optical measurement surface. After measurement the sample is wiped off the measurement surface with a lint free lab wipe. Innovation Applied ClipTips Thermo Scientific ClipTip pipette tips provide security with a unique and innovative interlocking technology that ensures a complete seal on every channel with minimal tip attachment and ejection force. Achieve newfound confidence knowing that once attached, your tips are locked firmly in place, and will not loosen or fall off regardless of application pressure. The innovative three interlocking clip design ensures the tip is held securely on the F1-ClipTip pipette until—and only until—it is released. Thermo Scientific Water Thermo Scientific Water is a complete line of water purification technologies which includes solutions for your most critical and everyday application needs, from electrodeionisation to reverse osmosis and distillation. Our water purification portfolio features advanced ergonomics and technology, including remote dispensing, UV intensity monitoring, small footprints and flexible dispensing options to provide a configuration that best suits your lab. Many water systems can be easily upgraded to allow for additional capacity. Thermo Scientific Mass Spectrometric Immunoassay (MSIA) Tips Thermo Scientific MSIA Tips are the next generation immunoaffinity approach that simplify peptide/ protein enrichment for downstream quantification using mass spectrometers. Mass spectrometry has become an important tool for biomarker research because of its sensitivity, accuracy and capability of resolving post-translational modifications (PTMs). Many protein biomarkers in biological samples are present in low concentrations (picograms/mL) and it is necessary to concentrate and enrich them for downstream mass spectrometric analysis. Immuno-enrichment is commonly employed; however, most conventional methods are laborius and not able to deliver reproducible enrichment of biomarker expressed in such low concentrations. MSIA tips provide a simple and effective way to enrich and concentrate target proteins down to femtomole level. Technology Advanced KingFisher FLEX Thermo Scientific™ KingFisher Flex system offers highly versatile, automated magnetic particle processing for DNA/RNA, protein or cell purification from virtually any source. Using the revolutionary magnetic particle separation technology, KingFisher Flex provides the fastest and easiest way for sample preparation from variety of sample material with excellent reproducibility and quality. Thermo Scientific KingFisher Kits complete the unique nucleic acid purification workflow, providing an optimised high-throughput method for extreme flexibility. Thermo Scienitific Pierce 660nm Quantitation kit The Pierce 660nm Protein Assay is a quick, ready-to-use colorimetric method for measuring protein concentration. The assay is more linear than coomassie-based Bradford assays and compatible with higher concentrations of most detergents, reducing agents and other commonly used reagents. The accessory Ionic Detergent Compatibility Reagent (IDCR) provides for even broader detergent compatibility, making the Pierce 660nm Protein Assay the only protein assay that is suitable for samples containing Laemmli SDS sample buffer with bromophenol blue. conditions between experiments. MagJet Extraction Kits MagJET nucleic acid purification kits utilise magnetic bead-based technology allowing for highly efficient nucleic acid isolation from a variety of samples at any throughput. MagJET kits provide high-purity DNA and RNA ready to use in routine and demanding downstream applications. The proprietary high capacity paramagnetic particles are optimised to isolate nucleic acids with superior purity and yields compared to other kits on the market. Pierce Magnetic Bead Kits for Protein Purification Thermo Scientific Pierce Magnetic Beads are specially designed and validated for use with automated magnetic particle processors. We offer glutathione and titanium dioxide magnetic beads for reliable and quick sample purification of the respective affinity targets. The high-performance, iron oxide, superparamagnetic particles are validated and optimised for use with high-throughput magnetic platforms, such as the Thermo Scientific KingFisher Flex Magnetic Particle Processor. Solutions Delivered Filter Units - Now Stem Cell Tested Thermo Scientific Nalgene Rapid-Flow disposable Filter Units with PES (polyethersulfone) membrane provide the last line of defense against cell culture contamination. Now you can guard your precious samples against contamination, and safely culture embryonic stem cells using filtered media - as long as you have the right filters and membranes! Our recent study (Culturing embryonic stem cells using media filtered with Thermo Scientific Nalgene Rapid-Flow PES filter units) shows that Embryonic Stem Cells grown in media filtered through Thermo Scientific Nalgene Rapid-Flow PES filters maintain normal growth and pluripotency, all without removing critical media components or adding deleterious compounds during the filtration process. Thermo Scientific Evolution 200 Spectrophotometers Perform experiments the way you want with the Thermo Scientific Evolution 201 and 220 spectrophotometers. Powerful software, cutting-edge technology and an array of accessories consistently deliver the high-quality results you expect. By using the same innovative software for on-board and computer control, your instrument is always up to date and ready for the next challenge. Conical Centrifuge Tubes Premium, high quality conical tubes that are environmentally friendly, offer the highest cleanliness with a recyclable, plastic rack, and allow for increased traceability with the largest writing area on the market. The Thermo Scientific Nunc tubes are the highest speed rated tubes on the market with our 15ml tubes rated to 10,500xg and the 50ml tubes rated to 17,000xg. Thermo Scientific Nunc Labware Products are made from high purity resins, and molded using our state-of-the-art processes. Plastic labware is a safer alternative to glass without sacrificing accuracy. Culturing embryonic stem cells culturing embryonic Maintaining pluripotent cells in culture represents a unique challenge for stem cell researchers. The culture system has to be closely controlled since any changes may easily trigger spontaneous differentiation or cell death. Growth factors, such as the leukaemia inhibitory factor (LIF) normally expressed in the trophoectoderm of the developing embryo, are often added to the culture media to promote long-term maintenance and prevent unwanted differentiation of pluripotent stem cells. The cost of these media supplements is often high, but they are critical in maintaining the pluripotency of cells. Therefore, it is important to ensure that the growth factors are maintained in the growth media at the proper concentrations. Growth media for cell culture is often sterile filtered in an effort to minimise the risk of contamination. Polyethersulfone (PES) membrane filters are used for this purpose due to the low protein binding and low extractable properties of the material. Filtering media for highly sensitive stem cells, however, introduces the possibility of removing important growth factors or adding compounds from the filter that may adversely affect the culture. To avoid this, many researchers add the most critical components (e.g. LIF) to the media after filtering. While this preserves the integrity of these components, it can be a vehicle for contamination. Here we show that filtering stem cell growth media using Thermo Scientific Nalgene Rapid-Flow PES filter units does not remove substantial amounts of LIF, nor does it add compounds that impair the growth of cells. We also show filtering the complete stem cell growth media using Nalgene Rapid-Flow PES filter units does not adversely affect stem cells, allowing them to maintain their normal growth and pluripotency characteristics. 26 Methods Mouse LIF ELISA All reagents were prepared according to the instructions for use of the Mouse LIF ELISA Quantikine assay kit (R&D Systems). 1500mL of Mouse ESC complete media was prepared and divided into three 500mL aliquots. Each aliquot was filtered through ten 500mL Rapid-Flow units of the same lot, and a sample of media was removed for testing after each filtration. Three different lots of the Rapid-Flow filters were used. New filter units were used for each filtration. Media samples were diluted 1:4 with PBS and tested according to LIF ELISA kit instructions. Plates were read on the Thermo Scientific Varioskan at 450nm. A reference reading at 570nm was subtracted from the test absorbance to eliminate background absorbance. The corrected absorbencies of the calibrator series were used to create a calibration curve with an R2 value of 0.99. Linear regression analysis was then used to calculate the LIF concentration of the unknown samples. Mouse Embryo Assay (MEA) Mouse embryo assay testing was conducted using an independent third-party Quality Control testing laboratory. Briefly, three 50mL batches of embryo culture media were filtered through Rapid-Flow filters from three different lots. Four 0.7μL droplets of filtered media were placed in the wells of a Nunc IVF multidish, and 21 embryos were cultured in the media for each filter lot. 15 embryos were cultured separately in unfiltered media as controls. Embryos were monitored for Culturing embryonic stem cells 96 hours to determine their progression to blastocyst stage. Embryos were scored after this time, and a result greater than 70% of embryos progressing to blastocyst stage was considered a passed test. Stem Cell Culture Stem cells were obtained from an established pluripotent culture of mESC. Seven batches of mESC growth medium were prepared and sterile filtered prior to adding LIF. After adding LIF, 3 of the batches were filtered once using Rapid-Flow filter units of 3 different lots. An additional 3 batches of medium were filtered five times using Rapid-Flow filter units of 3 different lots. Each filtration was performed using a new filter unit. mESC were seeded on Nunc 6-well Multidishes with mitotically inactivated Mouse Embryonic Fibroblast (MEF) feeder cells previously seeded in MEF-specific growth media. MEF media was exchanged with test media upon mESC seeding. Cultures were maintained on 6-well dishes with MEFs through five passages. Cells were grown two days between passages and media was changed on each day between passages. All feeding and passaging was performed with the proper medium batch corresponding to the test condition. Bright field photographs were taken in the fifth passage of cells on 6-well Multidishes. Also on the fifth passage, an additional Nunc 96-well optical bottom plate was cultured with mESC for approximately thirty hours before immunostaining of pluripotency markers. Immunocytochemistry Immunocytochemistry was performed in a Nunc 96-well optical bottom plate with a MEF feeder layer. Cells were fixed Figure 1. with 4% paraformaldehyde solution for 15 minutes. Cells were rinsed three times with DPBS and were blocked with 10% goat serum diluted in DPBST (DPBS with 0.1% TX-100) for 30 minutes at room temperature. Primary antibodies, SSEA-1, Oct-4, and Sox2 were diluted 1:400 in blocking solution and were added for an overnight incubation at 2-8°C. The cells were rinsed with DPBST three times and the appropriate secondary antibody (Alexa-488-gAM IgG, Alexa 488-gAR IgG, and Alexa 555-gAM IgM) diluted 1:1000 in blocking solution were incubated for one hour at room temperature. Cells were rinsed once with DPBST and DAPI diluted 1:1000 in DPBS was incubated for 5 minutes at room temperature. Cells were rinsed two times with DPBST followed by two washes with DPBS. The cells were stored in the second DPBS wash in the dark at 2-8°C until image analysis was completed. Bright field and fluorescent images were visualised with a Zeiss Axiovert 200 microscope and images were acquired with a Photometrics CoolSNAP ES2 camera. Image analysis was completed with AxioVision software. Results and Discussion Nalgene Rapid-Flow PES filters do not diminish LIF content in the mESC growth media The amount of LIF in mESC complete media was measured following successive filtration. ELISA results indicate that one time filtration did not appreciably affect the LIF concentration (Figure 1). Even over the course of ten filtrations, no more than 5% of LIF in the growth media is lost to filtration (Figure 1). Nalgene Rapid-Flow PES filters do not add deleterious compounds to the filtered media The highly sensitive Mouse Embryo Assay was conducted to assess potential harmful additions to the culture media from the filtration process. For two out of three tested filter lots, 95% of mouse embryos tested grew to blastocyst stage by 96 hours. For the remaining filter lot, 100% of the embryos progressed to blastocyst stage after 96 hours. These results indicate that filtering of media using Nalgene Rapid Flow PES filters does not add any deleterious compounds to the media that will affect cell growth Figure 1. The retention of LIF in the complete mESC growth media following multiple filtrations using Nalgene Rapid-Flow PES filters. Filtration did not appreciably affect LIF concentration. 27 Culturing embryonic stem cells Figure 2. Oct-4 SSEA -1 Sox2 Bright field 1 Filtration Figure 2. Immunofluorescence staining of pluripotent markers on mESC after being cultured for 5 passages in filtered media. 1 Filtration = media filtered once after formulation; 5 Filtrations = media filtered 5 times after formulation; and Control = media not filtered. Cells were counterstained with DAPI nuclear stain. mESC cultured in filtered media Control 5 Filtrations yielded comparable results to unfiltered controls. Media filtered with Nalgene Rapid-Flow PES filters support mESC growth and pluripotency Mouse embryonic stem cells were cultured on feeder cells through five passages using filtered medium. The mESC proliferated well throughout the span of the culture and displayed normal ESC morphology (Figure 2, bright field). The mESC pluripotency was evaluated by immunofluorescence staining of SSEA-1, OCT-4, and Sox2 markers (Figure 2). In both bright field and immunostained conditions, mESC cultured in filtered media yield comparable results as that of the control. These results indicate that mESC pluripotency was maintained throughout the time in culture with filtered medium containing LIF. Discussion Multiple media filtrations were conducted during this study in order to magnify any possible effect the filtration may have. For each successive filtration a new filter unit was used so that any compound that may have been removed by filtration would be truly eliminated, and any compound that may have been added by the filter would continue to be added as new filters 28 were used. Such stringent conditions are unlikely under normal application usage, however such a study ensures that any minimal effect that filtering may have become apparent during experimentation. Despite the extreme conditions posed by multiple media filtrations, embryonic stem cells continued to grow well and maintain pluripotency throughout the passages. These results indicate Nalgene Rapid-Flow PES filters are a safe solution for maintaining sterility in stem cell cultures. Conclusion • Nalgene Rapid-Flow filter units do not retain nor add components to the filtered media. • Sterile filtration of complete ESC growth media using Nalgene Rapid-Flow filter units does not adversely affect growth or pluripotency of the stem cells. • Nalgene Rapid-Flow PES filter units are a safe solution for maintaining sterility in stem cell cultures. Fits In. Introducing the NEW! range of Thermo Scientific Small Benchtop Centrifuges. Designed to fit your space & your applications. Developed with the flexibility to adapt with evolving clinical and research needs. It stands out with innovative technologies, anticipating and addressing your most pressing lab needs. Even those you don’t know you have yet. Stands Out. © 2013 Thermo Fisher Scientific Inc. All rights reserved. • To find out more visit thermofisher.com.au or call 1300-735-292 Differences in bacterial optical density measurements between spectrophotometers Brian C. Matlock, Richard W. Beringer , David L. Ash, Andrew F. Page, Thermo Fisher Scientific, Wilmington, DE, USA Michael W. Allen, Thermo Fisher Scientific, Madison, WI, USA Optical density (OD) measurement of bacterial cultures is a common technique used in microbiology. While researchers have relied on UV-Visible spectrophotometers to make these measurements, the measurement is actually based on the amount of light scattered by the culture rather than the amount of light absorbed. In their standard configuration, spectrophotometers are not optimised for light scattering measurements; commonly resulting in differences in measured absorbance between instruments. The standard phases of bacterial culture growth (lag, log, stationary, and death) are well documented, with the log phase recognised as the point where bacteria divide as rapidly as possible1. Using a spectrophotometer to measure the optical density at 600nm (OD600) of a bacterial culture to monitor bacterial growth has always been a central technique in microbiology. Three of the more common applications where bacterial OD600 is used are the following: (a) determination and standardisation of the optimal time to induce a culture during bacterial protein expression protocols, (b) determination and standardisation of the inoculum concentration for minimum inhibitory concentration (MIC) experiments, and (c) determination of the optimal time at which to harvest and prepare competent cells. Researchers continue to rely on absorbance spectrophotometers to make these OD 30 measurements. Optical density, however, is not a measure of absorbance, but rather a measure of the light scattered by the bacterial suspension which manifests itself as absorbance. The effect that the optical configuration of a spectrophotometer has on optical density measurements has been well documented.2-4 (Refer to Figure 1.) Instruments with different optical configurations will measure different optical densities for the same bacterial suspension. Differences in the optical configuration of the spectrophotometer make the largest contribution to the observed differences. Forward optical systems employ monochromatic light for the measurement of absorbance, where reverse optical systems utilise polychromatic radiation that is discriminated into individual wavelengths after it Differences in bacterial optical density measurements between spectrophotometers Spectrophotometers Thermo Scientific UV-Visible spectrophotometers used are described on Table 1. Growth Curve A 50 mL overnight E. coli JM109 culture was grown in a 250 mL baffled flask (16 hr , 250 rpm, 37°C) in Luria Bertani (LB) Broth. A batch culture was prepared by transferring 12 mL of the overnight culture in 600 mL of pre-warmed (37°C) LB media in a 2 L baffled flask. The batch culture was grown for a total of 9.5 hours. Culture Sampling All spectrophotometers were blanked using LB broth. Every 30 minutes, a 5 mL aliquot was sampled from the batch culture. A 3 mL aliquot of undiluted culture was transferred to a 10 mm cuvette, and the optical density at 600nm was measured on all the instruments listed in Table 1. A second 10 mm cuvette was prepared with an aliquot of the batch culture diluted in LB medium to an OD600 of approximately 0.5. This second OD measurement was measured to ensure that the optical density of the culture remained within the dynamic OD range of the spectrophotometers. Figure 1A is passed through the sample. (Refer to Figure 2.) Some components of the forward optical systems contribute to a difference in measured OD values: (a) the distance between sample and detector, (b) the size and focal length of any collector lens used, and (c) the area and sensitivity of the detector5. Despite current knowledge that different optical configurations will give different OD values, researchers continue to raise concerns about the differences in OD values seen among different spectrophotometers. In this study, we analysed OD values obtained from several spectrophotometers possessing various optical configurations. E. coli JM109 was grown in batch culture and the OD of the culture was monitored for 9.5 hours. Our measurements demonstrate the need to dilute cultures before measurement and show how to apply a conversion factor to the OD data in order to normalise differences in OD values due to the optical geometries found in different spectrophotometers. Figure 1. Figure 1: Light scattering in spectrophotometry. (A) In a non-scattering sample, the attenuation in light transmission between the light source and the detector is caused by the absorbance of light by the sample. (B) In a scattering sample, (i.e., a bacterial suspension), the light reaching the detector is further reduced by the scattering of light. This decrease in light reaching the detector creates the illusion of an increase in sample absorbance. Figure 2. Figure 2: Differences between forward and reverse optical systems. (A) In a forward optical system monochromatic light passes through the sample and then onto a detector. (B) In a reverse optical system, polychromatic light passes through the sample, discriminated into individual wavelengths and measured on an array detector. MATERIALS AND METHODS Strain E. coli JM109-endA1, recA1, gyrA96, thi, hsdR17 (rk–, mk-), relA1, supE44, (lac-proAB), [F′traD36, proAB, laqIq Z M15] (Promega, L2001) 31 Differences in bacterial optical density measurements between spectrophotometers Table 1: Thermo Scientific spectrophotometers Instrument Optical System Optical Geometry Light Source(s) Detector Thermo Scientific Evolution Array Reverse Optic – Deuterium and Tungsten Lamps Photodiode Array Thermo Scientific NanoDrop 2000c Reverse Optic – Xenon Flashlamp CMOS Array Thermo Scientific SPECTRONIC 200 Reverse Optic – Tungsten Lamp CCD Array Thermo Scientific Evolution 260 Bio Forward Optic Double Beam Xenon Flashlamp Silicon Photodiodes Thermo Scientific Evolution 300 Forward Optic Double Beam Xenon Flashlamp Silicon Photodiodes Thermo Scientific BioMate 3S Forward Optic Dual Beam Xenon Flashlamp Silicon Photodiodes Viable Counts At each 30 minute interval, an aliquot of the sample was also used to perform serial dilutions. Dilutions were plated on LB Agar plates and incubated at 37°C overnight. Colonies were counted to determine bacterial cell count (CFU/mL) at each time point. Figure 3: Comparison of growth curves of E. coli JM109 defined by measuring OD600 of diluted or undiluted bacterial samples. OD600 measurements were performed on the Evolution 260 Bio and the Evolution Array instruments. OD measurements were carried out every 30 minutes for 9.5 hours. Blue lines represent the OD600 from diluted culture samples. Samples were diluted so they were within the dynamic range of the optical system. Green lines represent the OD600 of undiluted culture samples. Figure 3. McFarland Standards OD600 measurements of aliquots from four McFarland standards (Remel, R20421, 1.0 – 4.0) were measured on each instrument (Table 1) by using 1 cm pathlength cuvettes. RESULTS OD600 data collected was from the undiluted and diluted cultures and representative growth curves are shown in Figure 3. OD600 values from diluted solutions were multiplied by the dilution factor and compared to undiluted samples. Divergence of the two plots illustrates that different optical configurations will have different dynamic ranges with respect to optical density measurements. When the corrected OD values for each spectrophotometer and the cell counts were compared, we found that instruments with similar optical systems produced similar OD curves over time (Figure 4). The BioMate™3S, which has a forward optical system but a dual-beam optical geometry, shows a systematically lower OD600 measurement than indicated by the plate counting method, most likely due to its unique optical geometry. The McFarland data shows a similar trend as was observed with the E. coli growth curves; similar optical systems grouped together (Figure 5). Conversion between Spectrophotometers The variation in optical density observed between two instruments can make standardisation of a protocol difficult. This is especially true when one laboratory tries to replicate data from another lab, but uses a different spectrophotometer, or when an aging spectrophotometer is replaced in the same lab. 32 Differences in bacterial optical density measurements between spectrophotometers Figure 6 shows the OD of a culture measured on both an Evolution™ 260 Bio and a BioMate 3S. The OD ratio between the two instruments was calculated at each time point. The OD600 ratio from each time point was determined and the average ratio was calculated and used as a multiplication factor. For the BioMate 3S, the calculated conversion factor was 1.46. Application of this correction factor to the OD data normalises the data and facilitates comparison between both instruments. To better facilitate agreement among spectrophotometers, a correction factor is included in the local control software of the BioMate 3S spectrophotometer. CONCLUSION It is critical to determine the optical density performance and dynamic range for your spectrophotometer when calculating OD measurements. For the most dynamic information about the growth curve it is important to dilute the cell suspension. OD measurements are highly dependent upon the optical system and geometry. Spectrophotometers with different optical systems and configurations will read different OD values for the same suspension. For example, there is substantial divergence between the reverse optical system Evolution Array™ and the forward optical system, dual-beam BioMate 3S. Finally, we present how to calculate a conversion factor that can be used to normalise the OD data so that appropriate comparisons can be made between spectrophotometers. It is important to note that this conversion factor is specific to a particular organism because the size and shape of the particle will affect the conversion factor . Figure 7: Equation Calculation for conversion factor between spectrophotometers. For the most accurate conversion factor two things are important; (1) All OD measurements used to calculate these values are from the corrected OD data (Figure 3), (2) take the average multiple conversion factor that are calculated across a range of various OD measurements. Figure 4. Figure 4: Growth curves of E. coli JM109 obtained from corrected OD values. The OD600 was measured on all instruments every 30 minutes for 9.5 hours. The OD600 data was corrected by the dilution factor used for OD measurement. Samples were diluted so they were within the linear range of the optical system. The corrected OD for each spectrophotometer was then plotted alongside the viable cell count data. Figure 5. Figure 6. Figure 5: The OD600 of various McFarland standards measured on spectrophotometers listed in Table 1 Figure 6: Application of a conversion factor to compare OD600 data from two different spectrophotometers. Green = original BioMate 3S data; Red = BioMate 3S data × factor of 1.46. References 1. Daniel R. Caldwell, Microbial Physiology and Metabolism (Wm. C.Brown Publishers, 1995), 55-59 2. Arthur L. Koch, “Turbidity Measurements of Bacterial Cultures in Some Available Commercial Instruments,” Analytical Biochemistry 38 (1970): 252-59 3. Arthur L. Koch, “Theory of the Angular Dependence of Light Scattered by Bacteria and Similar-sized Biological Objects,” Journal of Theoretical Biology 18 (1968): 133-56 4. J.V. Lawrence and S. Maier , “Correction for the Inherent Error in Optical Density Readings,” Applied and Environmental Microbiology Feb. (1977): 482-84 5. Gordon Bain, “Measuring Turbid Samples and Turbidity with UV-Visible Spectrophotometers,” Thermo Scientific Technical Note 51811 33 Fast purification of proteins with magnetic beads fast purification of proteins with magnetic beads Speed and sample purity are major challenges for proteomics researchers. Additionally, the amount of work required for proteomic analysis necessitates higher sample- and data-processing throughput. By combing an automated platform, such as the Thermo Scientific KingFisher Flex and Pierce Protein Magnetic purification kits, these challenges can be met. The KingFisher Flex Instrument can purify proteins ranging from simple recombinant proteins to complex therapeutic antibodies. Based on state-of-the-art magnetic separation technology, KingFisher systems enable you to process protein samples from virtually any source, including blood, cell cultures, tissue lysates and soil. Efficient operational speeds allow you to process up to 96 samples in as few as 15 minutes. The magnetic separation technology solves the problem of purifying recombinant proteins with highly complex structures and varying physiochemical properties. The Pierce Magnetic Beads are available with high-quality Streptavidin, Protein A, Protein G, glutathione and titanium dioxide for reliable and quick sample analysis. Together, 34 the KingFisher Flex instrument and the Pierce Magnetic Beads enable routine purification of antibodies, antigens, pharmaceuticals and vaccines using any kind of host cell. The automated fractionation is a valuable method for mass spectrometry analysis and biomarker discovery in which large numbers of patient samples are analysed for reliable detection of disease-specific peptides or proteins. The Protein A magnetic beads and Protein G magnetic beads are typically used for isolating antibodies from serum, cell culture supernatant or ascites and for immunoprecipitating antigens from cell or tissue extracts. The Glutathione magnetic beads purify glutathione S-transferase (GST)-fusion proteins from crude cell lysate prepared from bacteria, yeast or mammalian cells. The magnetic TiO2 Phosphopeptide Enrichment Kit Fast purification of proteins with magnetic beads Table 1. Applications for Thermo Scientific Pierce Magnetic Beads. Thermo Scientific Pierce Magnetic Bead Application and Sample Source Recommended Detection Methods Protein A • Purify antibodies from serum, cell culture supernatant or ascites • Immunoprecipitate antigens from cell or tissue extracts • SDS-PAGE gels • Western blot Protein G • Purify antibodies from serum, cell culture supernatant or ascites • Immunoprecipitate antigens from cell or tissue extracts • SDS-PAGE • Western blot • Mass spectrometry Glutathione • Purify GST-fusion proteins from crude cell lysate prepared from bacteria, yeast, plant or mammalian cells • SDS-PAGE • Western Blot • Mass Spectrometry Titanium Dioxide (Kit) • Isolate and enrich phosphopeptides from complex biological samples • Mass spectrometry is for isolating phosphopeptides from complex biological samples using titanium dioxide-coated magnetic beads. The isolated phosphopeptides are analysed downstream by mass spectrometry. Fast and convenient protocols are available for both manual and automated processing using each of the magnetic beads (Table 1). Methods Serum IgG purification: Approximately 0.5 mg of Pierce Protein A Magnetic Beads were added to 16 wells of a Deep Well 96 Plate. IgG was purified from rabbit serum using the “Antibody Purification” protocol. Briefly, beads were washed in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST), incubated 1 hour with 5 mg rabbit serum per well, washed in TBST and eluted in 0.1 M glycine, pH 2.8. Eluates were resolved by SDS-PAGE and stained with Thermo Scientific Imperial Protein Stain (Product No. 24615). (Figure 1.) Immunoprecipitation (IP) of Grp94: MOPC cell lysate (0.75 µg per sample) was combined with and without anti-Grp94 antibody (10 µg) and incubated overnight at 4˚C. Isolation of the Grp94/antibody pair was performed on the KingFisher Instrument using the “Immunoprecipitation Heated Elution” protocol. Briefly, Pierce Protein G Magnetic Beads (0.5 mg or 0.75 mg per well) were added to a Deep Well 96 Plate and washed with TBST. The antigen sample/antibody mixture was incubated for 1 hour with the beads. The beads were washed and eluted for 10 minutes at 96˚C with SDS- PAGE 35 Fast purification of proteins with magnetic beads Figure 1. Serum IgG purification with Thermo Scientific Pierce Protein A Magnetic Beads. IgG was purified from 5 mg of rabbit serum in 16 wells of a 96-well plate on a KingFisher instrument using 0.5 mg of magnetic beads per well. The diluted samples were resolved by SDS-PAGE and stained with Thermo Scientific Imperial Protein Stain. Figure 2. Manual vs. automated Grp94 immunoprecipitation. Panel A: Grp94 was isolated from MOPC cells using manual and automated protocols. Eluates prepared from 0.5 and 0.75 mg of Pierce Protein G Magnetic Beads were analysed by SDS-PAGE. The negative controls (no IP antibody) were prepared with 0.5 mg of bead. Panel B: MS/MS spectrum of a representative peptide (GVVDSDDLPLNVSR) of Grp94 identified from a band excised from the gel in Panel A. Digested protein was analysed with an LTQ XL Mass Spectrometer and Grp94 was identified with 39% sequence. Figure 3. Purification of GST-Rabaptin using glutathione beads. GST-Rabaptin was purified from bacterial lysate using 100 µl of Pierce Glutathione Magnetic Beads on a KingFisher instrument. All eluates were normalised by volume and analysed by SDS-PAGE. 36 Figure 1. Figure 2. Figure 3. reducing sample buffer. Alternatively, the same procedure was performed manually using a magnetic stand and microcentrifuge tubes. Eluates were resolved by SDS-PAGE and stained with Imperial Protein Stain. The Grp94 gel band was excised from the gel, digested with trypsin and analysed on a Thermo Scientific LTQ XL Mass Spectrometer. (Figure 2.) GST-Rabaptin Purification: Pierce Glutathione Magnetic Beads, 100 µL per well were added to 13 wells of a Deep Well 96 Plate with Binding/Wash Buffer (125 mM Tris, 150mM NaCl, pH 8.0) to a final volume of 200 µL. Protein purification was performed using the “GST Protein Purification” protocol. Briefly, beads were washed in Binding/Wash Buffer and incubated 1 hour with bacterial cell lysate. The beads were washed and eluted with 50 mM reduced glutathione prepared in Binding/ Wash Buffer. Eluates were resolved by SDS-PAGE and stained with Imperial Protein Stain. (Figure 3.) Phosphopeptide Enrichment from PBMCs: A tryptic digest of lymphocytes was processed using the Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment Kit. Samples were prepared using the “Phosphopeptide Enrichment-Deep Well” protocol on a KingFisher Instrument. Briefly, 100 µL of TiO2 magnetic beads were added to a Deep Well 96 Plate, rinsed with Binding Buffer and bound to 2 mg of peptide digest per well. The beads were washed and eluted. Eluted samples were dried, rehydrated in 5% acetonitrile/1% formic acid in water and injected into a Thermo Scientific LTQ-FT ultra highresolution mass spectrometer. (Figure 4. & Table 2.) Fast purification of proteins with magnetic beads Figure 4. Results and Discussion Pierce Magnetic Beads were tested in a variety of applications to demonstrate their utility in proteomics workflows using the KingFisher instrument. Rabbit serum IgG was purified using Pierce Protein A Magnetic Beads with excellent reproducibility across 16 wells (Figure 1). Samples were also processed in an additional 32 wells with similar results (data not shown). Figure 4. High-resolution LC-MS/MS data obtained from 2 mg of a tryptic peptide digest prepared from peripheral blood mononuclear cells (lymphocytes) enriched with the Thermo Scientific Pierce TiO2 Phosphopeptide Enrichment Kit. Peptide samples were processed on a KingFisher 96 Instrument. Panel A. Full scan chromatogram obtained on an LTQ-FT Ultra High-Resolution Mass Spectrometer, resolution 200K, mass accuracy < 2 ppm. Panel B. Zoom of one full scan, retention time 64.50 minutes. Panel C. MS2 fragmentation spectrum of single phosphopeptide ion, parent mass 1582.70. Peptide sequence: SS' PFKVS' PLTFGR. The protein was identified as serum deprivation response protein. Panel D. MS2 fragmentation spectrum of a single phosphopeptide ion, parent mass 1722.80. The peptide sequence is LPS' GSGAASPTGSAVDIR. The protein was identified as AHNAK nucleoprotein isoform 1. (' = site of phosphorylation). Table 2. Summary of the Figure 4 data obtained from sample enriched & not enriched with the Pierce TiO2 Phosphopeptide Enrichment Kit Enriched Non-Enriched Total number of proteins identified 185 247 Total number of phosphoproteins identified 160 1 Total number of peptides identified 28347 28457 Total number of phosphopeptides identified 28009 7 Total number of unique phosphopeptides identified 177 1 Relative enrichment for phosphopeptides (%) 86 0.3 References 1 Thermo Fisher Scientific, Rockford, Ill., USA 2 Biomarker Research Initiatives in Mass Spectrometry (BRIMS), Thermo Fisher Scientific, 790 Memorial Dr., Suite 201, Cambridge, MA 02139, USA 37 Benefits of low retention microcentrifuge tubes When working with highly viscous liquids or irreplaceable reagents within a microcentrifuge tube, a film can form around the inside tube wall resulting in liquid retention and reducing sample recovery. Depending on the retained volume and application, this can affect assay accuracy and precision. In this study, liquid retention levels of different types of solutions were measured in microcentrifuge tubes from major manufacturers. The data demonstrated that the Thermo Scientific™ Snap Cap Low Retention Microcentrifuge Tube retained less volume on average, resulting in improved accuracy and precision. benefits of low retention microcentrifuge tubes Why Use a Low Retention Microcentrifuge Tube? Liquid handling precision is becoming increasingly important in data consistency and reproducibility. Research methods such as real-time PCR, and mass spectrometry assist detection and quantification of biological and small molecules in low volumes. It is commonly known that surface tension of liquids to polypropylene varies amongst different types of solutions. For instance, liquids with low surface tension, such as detergents, tend to wet the surface of the polypropylene wall and form a film, compromising accuracy. Viscous solutions, including glycerol, often magnify liquid retention problems. Since the enzymes and PCR master mixes that are routinely used in molecular applications are stabilised and stored in the presence of glycerol, it is important to reduce liquid retention in order to maximise recovery. Thermo Scientific Snap Cap Low Retention Microcentrifuge Tubes are more hydrophobic than standard tubes. Unlike some low retention technologies available on the market, our approach does not utilise a coating or plastic imbedding process that could possibly leach, eliminating the danger of sample contamination and negatively affecting bio-assay results. Manufactured without the aid of plasticisers, Thermo Scientific Low Retention technology successfully minimises 38 the formation of liquid films during use, improving accuracy and precision. Further value is added for the recovery of expensive samples. Physical inspection and rigorous quality testing is a key component in manufacturing and consistently delivering a quality product that can be relied upon to meet and exceed your centrifugation needs. Materials and Methods Thermo Scientific Low Retention tubes with a working volume of 1.6 ml and three other competing products were used in a gravimetric assay conducted at an independent third-party testing facility. Green food dye (50% diluted) with deionised water was spiked into the following solutions: Table A. Solution A 50% glycerol spiked with 100 μl of diluted green food dye Solution B 40 μl/ml BSA spiked with 100 μl of diluted green food dye Solution C Water spiked with 100 μl of green food dye Each tube’s dry weight was recorded. 1.5 ml of Solution A was added to a sample size of Thermo Scientific tubes, as well as to tubes from Competitor A, Competitor B and Benefits of low retention microcentrifuge tubes Figure 1. Comparative chart of the overall calculated average of liquid volume. Competitor C. These tubes were weighed again and stored at room temperature for 24 hours. After 24 hours the tubes were visually inspected for any apparent visible liquid loss using the graduation marks on the tubes as the reference point. Solution A was then aspirated out of each tube. Comments regarding visible liquid retention were noted and then each tube was weighed. This procedure was repeated for Solution B and Solution C. Results and Discussion Table B. Overall liquid retention Overall ranking Tube tested/solution Overall 1 Thermo Scientific Low Retention Microcentrifuge Tube 0.00810 2 Competitor A 0.00820 3 Competitor B 0.00860 4 Competitor C 0.01890 Table B. Summarises the overall calculated average of liquid volume retained by each tube for the three solutions. Thermo Scientific microcentrifuge tubes had the lowest calculated liquid volume retained compared to the low retention tubes of Competitor A, Competitor B and Competitor C. This value is significant in that it is achieved without any secondary processes or additives to the plastic that could introduce an unknown factor into the experiment. Small differences in retained volumes can be significant when considering the costs of reagents.Thermo Scientific Snap Cap Low Retention Microcentrifuge Tubes performed best overall compared to products from other manufacturers. Versatile as a standard snap cap but with low retention capabilities, this product will be a cost effective, dependable component to your workflow solution. 39 Total protein quantitation total protein quantitation Total protein quantitation is a common measurement in life science, biopharmaceutical and food and beverage laboratories. Total protein quantitation is a common measurement in life science, biopharmaceutical and food and beverage laboratories. Some of the most frequently used assays for total protein quantitation are dye binding assays, such as the Coomassie-based Bradford Assay. Although it is widely used, the Bradford Assay has several disadvantages. Many substances, including some detergents, flavonoids and protein buffers are known to interfere with the colorimetric properties this method relies on. Additionally, the linearity of the Bradford Assay is limited in both quality and range. The broad substance compatibility and improved linearity of the Thermo Scientific Pierce 660nm Assay in combination with its simple, single-reagent format make it a more accurate and convenient method for many routine applications. When performed on a Thermo Scientific UV-Visible spectrophotometer with embedded BioTest software, such as the Evolution™ 60S, BioMate™ 3S or GENESYS™ 10S Bio, the local control interface eliminates the need for manual programming of the Pierce™ 660nm Protein Assay. Background The Thermo Scientific Pierce 660nm Protein Assay is a quick, ready-to-use colorimetric method for total protein quantitation. The assay is reproducible, rapid and more linear than the Bradford Assay. In addition, the Pierce 660nm Protein Assay is compatible with high concentrations of most commonly used reagents, such as detergents and reducing agents. As in a Bradford Assay, protein concentrations are estimated by 40 reference to a series of standard protein dilutions assayed alongside the unknown samples. Every total protein assay method exhibits some degree of varying response towards different proteins. These differences are related to variations in amino acid sequence, isoelectric point, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein’s colour response. Thus, the selection of an appropriate reference protein is the key to obtaining high quality results. The ideal protein to use as a reference standard in any protein assay is a purified preparation of the same protein that is to be measured in the sample. In the absence of such a reference protein, an alternative protein that produces a similar colour response to that of the sample protein may be used. The two most commonly used substitutes for a reference protein are Bovine Serum Albumin (BSA) and Bovine Gamma Globulin (BGG). BSA is a suitable standard when the sample contains primarily albumin, or the sample protein has a similar response to the dye as BSA. For a colour response that is typical of purified antibodies and most non-serum protein mixtures (e.g., cell lysates), BGG is an appropriate standard protein. About the Pierce 660nm Assay The Pierce 660nm Protein Assay uses a proprietary dye-metal complex which binds to protein in acidic conditions, causing a shift in the dye’s absorption maximum, which is measured at 660nm. The dye-metal complex is reddish-brown in colour, and turns green upon protein binding. This colour change is produced by deprotonation of the dye at low pH facilitated Nicole Kreuziger Keppy, Thermo Fisher Scientific, Madison, WI, USA by protein binding interactions between positively charged amino acid groups in the protein and the negatively charged deprotonated dye-metal complex.1 Consequently, the complex interacts primarily with basic residues in the protein, such as histidine, arginine, and lysine; and, to a lesser extent tyrosine, tryptophan and phenylalanine. The Pierce 660nm Protein Assay has a protein-to protein variation of 37%; however, it is more linear then the Bradford assay (Figure 1).2 Thus, it produces more accurate results when the appropriate standard is used. The linear detection range for the test tube method of the 660 assay is 25–2,000 µg/mL for BSA and 50–2,000 µg/mL for BGG. Figure 1. Total protein quantitation In addition, the colour produced in the assay is stable and increases in proportion to a broad range of increasing protein concentrations, even in the presence of detergents and reducing agents that would be incompatible with Bradford and BCA Protein Assays. The optional Ionic Detergent Compatibility Reagent (IDCR) may be added to the assay reagent to increase compatibility with high amounts of ionic detergents. This allows samples containing Laemmli SDS sample buffer with bromophenol blue to be measured. The IDCR completely dissolves by thorough mixing and does not have any affect on the assay. A list of maximal concentrations for substances known to be compatible with the Pierce 660nm Protein Assay can be found in the product’s instructions. Figure 2. Figure 1: Performance comparison of the Thermo Scientific Pierce 660nm Protein Assay vs. the Bio-Rad® Bradford Protein Assay. Both assays were performed according to the test-tube procedure using 100 µL of BSA. The Pierce 660nm Protein Assay has a greater linear range (25–2,000 µg) compared with the Bradford Assay (125–1,000 µg). Absorbance values were measured at 660nm for the 660nm Protein Assay and 595nm for the Bradford Protein Assay. Figure 2: Example of a Pierce 660 Protein Assay BSA Standard Curve obtained on a Thermo Scientific GENESYS 10S UV-Visible spectrophotometer 41 Total protein quantitation Experimental Method and Results The Pierce Pre-diluted Protein Assay Standards: Bovine Serum Albumin (BSA) Set was used to prepare a standard curve. The kit contains seven standardised BSA solutions at concentrations of 125, 250, 500, 750, 1000, 1500 and 2000 µg/mL. A 25 µg/mL standard was prepared by mixing 10 µL of the 1000 µg/mL BSA standard with 390 µL of a prepared 0.9% saline and 0.05% sodium azide buffer solution. Two BSA samples of unknown concentration were also prepared using the 0.9% saline and 0.05% sodium azide buffer solution, and the buffer solution was used as the blank. Following the provided 660 Assay instructions, 1.5 mL of assay reagent was added to 0.1 mL of each standard, sample and blank solution and incubated for five minutes. All solutions were read at a wavelength of 660nm on a Thermo Scientific GENESYS 10S UV-Vis spectrophotometer with a 1.8nm spectral bandwidth. The standard results, as shown in Figure 2, depict a linear curve fit with a correlation coefficient of 0.999, a clear indication that the Pierce 660nm Protein Assay is linear for concentration of 25– 2000 µg/mL. The sample results, as they are displayed n the local control software, are shown in Figure 3. Figure 3: Example of a Pierce 660 Assay BSA Sample results obtained on a Thermo Scientific GENESYS 10S UV-Visible spectrophotometer Figure 4: Example of the Protein Assay Menu screen on a Thermo Scientific BioMate 3S UV-Visible spectrophotometer References 1. U.S. Patent Application 20090197348, Pierce Biotechnology, Inc., 2009. 2. Pierce 660nm Protein Assay Instructions, Thermo Fisher Scientific, 2008. 42 Figure 3. Performing the 660 Assay using Local Control BioTest Software Embedded BioTest software in the local control interface of the Evolution 60S, BioMate 3S and GENESYS 10S Bio spectrophotometers provides pre-programmed life science assays, which includes the Pierce 660nm Protein Assay and eliminates the need for manual programming. When using one of these instruments, simply select Protein Assays from the BioTest menu, then select the Pierce 660nm Protein Assay from the list of assays shown in Figure 4 and follow the prompts. Summary As we can see from this data, the Pierce 660nm Protein Assay provides an easy-to-use, accurate and reliable method for total protein concentration measurements. In addition, combining the assay with a Thermo Scientific UV-Visible spectrophotometer with built-in BioTest software, such as the Evolution 60S, BioMate 3S and GENESYS 10S Bio, eliminates the need for manually programming the method. This simplifies the analytical process even further, saving time and reducing the potential for analyst errors. Figure 4. Confidence. Convenience Our Essential Support Plans give you confidence that your instruments will continually operate at optimum efficiency. You will appreciate having the priority attention of our expert engineers, whose main goal is to prevent disruptions and keep your laboratory operating efficiently. Eliminate the uncontrollable costs of unplanned maintenance and repairs. flexibility • To find out more visit thermofisher.com.au/service or call1300-736-767 43 Ultra low level seawater analysis As the world leader in serving science we are uniquely positioned to combine our unrivalled depth of product, application and service expertise with our extensive range of Scientific, Healthcare, Environmental and Industrial Process products to provide tailored solutions for each & every customer. Australia: For customer service, call 1300-735-292 Visit us online at: www.thermofisher.com.au New Zealand: For customer service, call 0800-933-966 Visit us online at: www.thermofisher.co.nz ©2013 Thermo Fisher Scientific Inc. All rights reserved. 44