Download Western blot Fast purification Comparative performance

Bio
08
Western blot
Detection using CCD digital imaging
Fast purification
of proteins with magnetic beads
Comparative
performance
Evaluation of MSIA™ & Magnetic
Bead Formats
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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
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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
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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
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43
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44