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Protease and Phosphatase Inhibitor Cocktails Pre-mixed formulations to preserve the integrity of your protein sample EMD Millipore is a division of Merck KGaA, Darmstadt, Germany Protease and Phosphatase Inhibitor Cocktails Introduction Ensure the integrity and modification state of EMD Millipore’s protease and phosphatase inhibitor purified proteins by using protease and phosphatase cocktails are provided as ready-to-use, no-waste inhibitor cocktails, highly specific protease inhibitors liquid stock solutions with complete formulation and PhosphoSafe™ Extraction Reagent in cell lysates details enclosed. Choose the product that’s designed and tissue extracts. Protease and phosphatase for your specific application and experience better inhibitors are essential for successful protein protein research results. expression and characterization, biomarker discovery, mapping of post-translational modifications, protein expression profiling, protein quantification, and proteomics research. Table of Contents Protease 03 page Inhibitor Cocktails 08 Phosphatase page Inhibitor Cocktails 2 Protease Inhibitor Cocktails Proper functioning of the cell requires a carefully controlled level of important structural, functional, and regulatory proteins. Hence, proteins are continuously synthesized and degraded in plant and animal cells and a finely tuned balance exists between their rate of synthesis and breakdown that determines the concentration of any given protein. Protein degradation is an essential process whereby damaged, unwanted, or “used” proteins are continuously eliminated. The extraordinary complexities of proteins in biological samples present a difficult challenge for their analysis. Hence, in a laboratory set up where the integrity of proteins is to be maintained during extraction, purification, and analysis the use of protease inhibitors is highly desired to minimize any non-specific protein breakdown. Protease inhibitor cocktails are important tools for scientists engaged in a variety of proteomics studies, including protein characterization, biomarker discovery, mapping of post-translational modifications, protein expression profiling, and the quantitative measurement of proteins. The addition of a protease inhibitor cocktail or protease inhibitor ensures the integrity of proteins for downstream analysis and further characterization of samples. Advantages • Convenient: Ready-to-use, waste-free liquid formulations ensure greater convenience • Consistent: Stringent quality control ensures reproducibility and excellent inhibition over a wide range of protease classes • Flexible: Full disclosure of formulations and a comprehensive selection of specific cocktails designed to inhibit proteolytic activity from most tissue or cell type extracts ensures greater experimental flexibility 3 Application-Specific Protease Inhibitor Cocktails EMD Millipore inhibitor cocktails are available in a variety of formulations and formats to ensure the best results on a wide range of starting materials and applications. For example, inhibitor cocktails are available without EDTA for purification schemes involving metal ion-chelating chromatography or analysis using two dimensional (2D) gel electrophoresis. Select formulations use recombinant aprotinin for applications that require the use of animal-free reagents. The table below summarizes the components and application specificity of each protease inhibitor cocktail. 4 Product Components Application Cat. No. Protease Inhibitor Cocktail Set I AEBSF, HCl; Aprotinin, Bovine Lung; E-64 Protease Inhibitor; EDTA, Disodium, Leupeptin, Hemisulfate General Use 80053-844 Protease Inhibitor Cocktail Set I, Animal-Free AEBSF, HCl; Aprotinin, Recombinant; E-64 Protease Inhibitor; EDTA, Disodium; Leupeptin Hemisulfate General Use and for applications which require the use of animal-free reagents. 80017-342 Protease Inhibitor Cocktail Set II AEBSF, HCl; Bestatin; E-64 Protease Inhibitor; EDTA, Disodium; Pepstatin A Bacterial cell extracts (except those intended for metal chelation chromatography) 80053-850 Protease Inhibitor Cocktail Set III, EDTA-Free AEBSF, HCl; Aprotinin, Bovine Lung; Bestatin; E-64 Protease Inhibitor; Leupeptin, Hemisulfate; Pepstatin A Mammalian cells and tissue extracts purified using metal chelation chromatography; samples to be analyzed by 2-D gel electrophoresis 80053-854 Protease Inhibitor Cocktail Set III, Animal-Free AEBSF, HCl; Aprotinin, Recombinant; Bestatin; E-64 Protease Inhibitor; Leupeptin, Hemisulfate; Pepstatin A Mammalian cells and tissue extracts and for applications which require the use of animalfree reagents 80017-246 Protease Inhibitor Cocktail Set IV AEBSF, HCl; E-64 Protease Inhibitor; Pepstatin A; o-Phenanthroline Fungal and yeast cell extracts 80503-258 Protease Inhibitor Cocktail Set V, EDTA Free AEBSF, HCl; Aprotinin , Bovine Lung; E-64 Protease Inhibitor; Leupeptin , Hemisulfate Mammalian cells and tissue extracts purified using metal chelation chromatography; samples to be analyzed by 2-D gel electrophoresis 80601-615 Protease Inhibitor Cocktail Set V, Animal-Free AEBSF, HCl; Aprotinin , Recombinant; E-64 Protease Inhibitor; Leupeptin , Hemisulfate Mammalian cells and tissue extracts and for applications which require the use of animalfree reagents 80017-350 Protease Inhibitor Cocktail Set VI AEBSF, HCl; Bestatin; E-64 Protease Inhibitor; Leupeptin, Hemisulfate; o-Phenanthroline; Pepstatin A Plant cell extracts 80602-622 Protease Inhibitor Cocktail Set VII AEBSF, HCl; Bestatin; E-64 Protease Inhibitor; Pepstatin A; Phosphoramidon, Disodium Salt Proteins containing His tag sequences 80602-626 Protease Inhibitor Cocktail Set VIII ALLN; Cathepsin Inhibitor I; E-64 Protease Inhibitor Broad range cysteine protease inhibition 80603-796 Serine Protease Inhibitor Cocktail Set I AEBSF, HCl; Aprotinin, Bovine Lung; Elastatinal, GGACK Broad range serine protease inhibition 80600-148 Protease Inhibitor Set AEBSF, HCl; E-64; EST; Leupeptin, Hemisulfate ; Pepstatin A; TLCK, HCl; TPCK For screening for suitable protease inhibitors 80053-842 Featured Protease Inhibitor Cocktail Protease Inhibitor Cocktail Set III, EDTA-Free Cat. No. 80053-854 Form: Liquid This cocktail is recommended for use with mammalian cells and tissue extracts and for bacterial cell extracts being used for metal ion chelation chromatography. Cocktail contains six protease inhibitors (in 1 mL DMSO) with broad specificity for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases. One mL is sufficient for about 20 g tissue. Note: Available as 1 x 1 mL or as a set of 5 x 1 mL. Protease Inhibitor Molecular Weight (MW) Concentration in Vial Target Proteases 239.5 100 mM Serine proteases Aprotinin, bovine lung 6512 80 µM Serine proteases and esterases Bestatin 308.4 5 mM Aminopeptidease B and leucine aminopeptidase E-64 Protease Inhibitor 357.4 1.5 mM Leupeptin, hemisulfate 475.6 2 mM Cysteine proteases and trypsin-like proteases Pepstatin A 685.9 1 mM Aspartic proteases AEBSF, hydrochloride Cysteine proteases More efficient than competitor product: Volume of cell extract 500 mL Required amount of EMD Millipore Protease Inhibitor Cocktail Set III Required amount of Supplier R Protease Inhibitor Cocktail 1 mL 10 tablets 5 Protease Inhibitors | Frequently Asked Questions What criteria should I use when selecting a protease inhibitor? When processing cells or tissues, you should assume that active proteases are present. Therefore, you should include protease inhibitors in the early steps of sample preparation. For best results, add protease inhibitors to the protein solution sample just before use to ensure the maximum activity for the cocktail during sample preparation. Typically, inhibitors should not be dissolved in buffers and stored for long periods of time. Since different cells and tissue types express different protease profiles, our application-specific cocktails are formulated with different sets of inhibitors. For example, serine proteases are widely distributed in all cells; bacterial cells contain higher levels of serine and metalloproteases; animal tissue extracts are rich in serine, cysteine, and metalloproteases, and plant extracts contain higher quantities of serine and cysteine proteases. Other selection criteria include what type of method is being used. EDTA is typically not desirable in 2-D gel electrophoresis studies. I currently use protease inhibitor cocktail tablets. Is it easy to switch to ready-to-use liquid cocktails? Yes, in fact, there are several benefits to using pre-made, liquid, protease inhibitor cocktails. In general, liquid cocktails are: • S table — our cocktails offer a greater degree of stability compared to tablets dissolved in medium (see Figure 1) • E fficient — the liquid cocktail format allows immediate exposure of inhibitors to proteases present in samples • F lexible, minimizing waste — you only need to add the amount of cocktail needed for your specific protocol •C onvenient, easy to use — working reagents are dissolved and ready to use •C onsistent — the same quantity of inhibitor is always dispensed I work in an automation lab and want to use the cocktails on a liquid handling workstation. Are Calbiochem® protease inhibitor cocktails stable? Our cocktails are compatible for use on liquid handlers and are stable, as shown in the data below. Calbiochem® Protease Inhibitors offer greater efficiency and stability Proteolytic Activity 70000 day 0 A = Competitor, 8 °C 60000 day 1 B = Calbiochem® Cocktail VII, 8 °C 50000 day 3 C = Competitor, room temp. 40000 day 5 D = Calbiochem® Cocktail VII, room temp 30000 20000 10000 0 6 No Inhibitors A B C D Figure 1. Stability of Protease Inhibitor Dilutions in BugBuster® Lysis Reagent Protease inhibitors were diluted to the prescribed working concentration (Competitor or Calbiochem® Cocktail VII, Cat. No. 80602-626). The ability of the inhibitors to inhibit the proteolytic activity of PRONASE® (Cat. No. 80602-612) was measured by using the Universal HT Protease Assay on days zero, one (24 h post dilution), three (72 h) and five (120 h). The Universal HT Protease Assay quantifies protease activity using a fluorescein thiocarbamoyl-casein derivative (FTCcasein). Proteolytic activity liberates FTC-labeled peptides, which results in enhanced fluorescence (Ex.max: 495 nm; Em.max: 525 nm). Addition of the protease inhibitor cocktails inhibits the proteolytic activity of the PRONASE® (Cat. No. 80602-612), resulting in reduced fluorescence. On day 1, for samples incubated at 8 °C, the competitor tablet inhibited the proteolytic activity by 50% and the Calbiochem® Cocktail VII inhibited the proteolytic activity by 70%. On day 5, for samples incubated at 8 °C, the competitor tablet caused a 29% decrease in proteolytic activity in comparison to the Calbiochem® cocktail VII, which caused a 57% decrease in proteolytic activity. The data show that the efficiency of the cocktail remained higher than the competitor’s tablet in this study. Key Products Description Cat. No. Protease Inhibitor Cocktail Set I 80053-844 Protease Inhibitor Cocktail Set I, Animal-Free 80017-342 Protease Inhibitor Cocktail Set II 80053-850 Protease Inhibitor Cocktail Set III, EDTA-Free 80053-854 Protease Inhibitor Cocktail Set III, Animal-Free 80017-246 Protease Inhibitor Cocktail Set IV 80503-258 Protease Inhibitor Cocktail Set V, EDTA-Free 80601-615 Protease Inhibitor Cocktail Set V, Animal-Free 80017-350 Protease Inhibitor Cocktail Set VI 80602-622 Protease Inhibitor Cocktail Set VII 80602-626 Protease Inhibitor Cocktail Set VIII 80603-796 Serine Protease Inhibitor Cocktail Set I 80600-148 Protease Inhibitor Set 80053-842 Protease Arrest™ Reagent 80510-638 7 Phosphatase Inhibitor Cocktails Phosphorylation and dephosphorylation of proteins mediate signal transduction events that control a multitude of cellular processes such as cell division, cell proliferation, and apoptosis. Target proteins are phosphorylated by protein kinases that transfer a phosphate group to a specific protein, typically at serine, threonine, or tyrosine residues. The covalent attachment of a phosphoryl group changes the conformation of the protein and alters its ability to interact with a ligand. The phosphate group is removed by the action of protein phosphatases, which restore the protein to its original dephosphorylated state. For accurate characterization of the structure and function of phosphorylated proteins, it is critical to preserve their phosphorylation state during extraction from cell and tissue lysates. Use our phosphatase inhibitor cocktails and PhosphoSafe™ Extraction Reagent to protect phosphoproteins from different families of phosphatases. Application-Specific Phosphatase Inhibitor Cocktails EMD Millipore inhibitor cocktails are available in a variety of formulations and formats to ensure the best results on a wide range of starting materials and applications. The table below summarizes the components and application specificity of each phosphatase inhibitor cocktail. Product Components Application Phosphatase Inhibitor Cocktail Set I (-)-p-Bromotetramisole oxalate; Cantharidin; Microcystin LR, Microcystis aeruginosa Protection against alkaline phosphatases and Ser/Thr phosphatases such as PP1 and PP2A. Note: Not available for sale outside of the United States 80501-128 Phosphatase Inhibitor Cocktail Set II Imidazole; Sodium Fluoride; Sodium Molybdate; Sodium Orthovanadate; Sodium Tartrate, dihydrate Protection against acid and alkaline phosphatases and protein tyrosine phosphatases (PTPs) 80501-130 Phosphatase Inhibitor Cocktail Set III Sodium Fluoride; Sodium Orthovanadate; Sodium Pyrophosphate, decahydrate; b-Glycerophosphate Protection against Ser/Thr phosphatases and protein tyrosine phosphatases (PTPs) 80017-332 Phosphatase Inhibitor Cocktail Set IV (-)-p-Bromotetramisole oxalate; Cantharidin; Calyculin A, Discodermia calyx Protection against alkaline phosphatases and Ser/Thr phosphatases such as PP1 and PP2A 101388-172 Protection against Ser/Thr phosphatases and Protein Tyrosine Phosphatases (PTPs) 82601-054 PhosphoSafe™ Extraction Reagent 8 Cat. No. Featured Phosphatase Inhibitor Cocktail Phosphatase Inhibitor Cocktail Set II Cat. No. 80501-130 Form: Liquid A cocktail of five phosphatase inhibitors for the inhibition of acid and alkaline phosphatases as well as protein tyrosine phosphatases (PTPs). Suitable for use with tissue and cell extracts, including extracts containing detergents. Each vial contains 1 mL aqueous solution of the phosphatase inhibitor cocktail. The concentrations of the individual inhibitors are shown in the table. Note: 1 set = 5 x 1 mL. Phosphatase Inhibitor Molecular Weight (MW) Concentration in Vial Target Proteases Imidazole 68.1 200 mM Alkaline phosphatase Sodium fluoride 42.0 100 mM Acid phosphatase Sodium molybdate 205.9 115 mM Acid phosphatase Sodium orthovanadate 183.9 100 mM Protein tyrosine phosphatase, alkaline phosphatase Sodium tartrate, dihydrate 230.1 400 mM Acid phosphatase More efficient than competitor product: Volume of cell extract 500 mL Required amount of EMD Millipore Phosphatase Inhibitor Cocktail Set III Required amount of Supplier R Phosphatase Inhibitor Cocktail 1 set (5 x 1 mL) 50 tablets Key Products Description Cat. No. Phosphatase Inhibitor Cocktail Set I 80501-128 Phosphatase Inhibitor Cocktail Set II 80501-130 Phosphatase Inhibitor Cocktail Set III 80017-332 Phosphatase Inhibitor Cocktail Set IV 101388-172 Phosphatase Inhibitor Cocktail Set V 82603-842 PhosphoSafe™ Extraction Reagent 82603-842 9 Related Products Description 80031-734 BugBuster® Master Mix Extraction reagent 82601-026 BugBuster® Plus Benzonase Nuclease 80108-830 BugBuster® Plus Lysonase™ Kit 80602-814 BugBuster® HT Protein Extraction Reagent 80500-206 BugBuster® 10X Protein Extraction Reagent 80500-202 BugBuster®(primary amine-free) Extraction Reagent 80508-842 PopCulture® Reagent 80600-006 Insect PopCulture® Reagent 80601-830 YeastBuster™ Protein Extraction Reagent 60801-624 CytoBuster™ Protein Extraction Reagent 80509-652 NucBuster™ Protein extraction Reagent 80601-632 ProteoExtract® Subcellular Protein Extraction Kit 80601-632 ProteoExtract® Native Membrane Protein Extraction Kit 80016-460 ProteoExtract® Transmembrane Protein Extraction Kit 80017-516 ProteoExtract® Complete Mammalian Protein Extraction Kit 80601-840 ProteoExtract® Cytosol/Mitochondria Fractionation Kit ProteoExtract® Tissue Dissociation Kit 10 Cat. No. BugBuster® Protein Extraction Reagent 80511-866 200066-520 ProteoExtract® All-in-One Trypsin Digestion Kit 80603-586 ProteoExtract® Phosphopeptide Enrichment TiO2 Kit 82603-358 ProteoExtract® Phosphopeptide Enrichment SCIMAC Kit 82603-360 11 www.emdmillipore.com BugBuster, PopCulture, ProteoExtract, Calbiochem, Amicon, and PRONASE are registered trademarks of Merck KGaA, Darmstadt, Germany. EMD Millipore, the M mark, YeastBuster, CytoBuster, NucBuster, PhosphoSafe, PureProteome, and Protease Arrest™ are trademarks of of Merck KGaA, Darmstadt, Germany. Lit No. PB4994EN00 LS-SBU-11-05727 Printed in the USA 02/2012 © 2012 EMD Millipore Corporation, Billerica, MA, USA. All rights reserved. 0212 3M Lit. No. 05105