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Protease and Phosphatase Inhibitor CocktailsPre-mixed formulations to preserve the integrity of your protein sample

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Introduction

Ensure the integrity and modification state of

purified proteins by using protease and phosphatase

inhibitor cocktails, highly specific protease inhibitors

and PhosphoSafe™ Extraction Reagent in cell lysates

and tissue extracts. Protease and phosphatase

inhibitors are essential for successful protein

expression and characterization, biomarker discovery,

mapping of post-translational modifications, protein

expression profiling, protein quantification, and

proteomics research.

EMD Millipore’s protease and phosphatase inhibitor

cocktails are provided as ready-to-use, no-waste

liquid stock solutions with complete formulation

details enclosed. Choose the product that’s designed

for your specific application and experience better

protein research results.

Table of Contents

Inhibitor Cocktails

Inhibitor Cocktails

Protease

Protein

Phosphatase

03

11

page

page

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08

Protease and Phosphatase Inhibitor Cocktails

Purification, Concentration and Buffer Exchange

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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.

Protease Inhibitor Cocktails

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

For our current pricing or to download comprehensive information on these products, visit www.emdbiosciences.com/proteaseinhibitors

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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.

Application-Specific Protease Inhibitor Cocktails

Product Components Application Catalogue No.

Protease Inhibitor Cocktail Set I

AEBSF, HCl; Aprotinin, Bovine Lung; E-64 Protease Inhibitor; EDTA, Disodium, Leupeptin, Hemisulfate

General Use 539131

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.

535142

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)

539132

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

539134

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 animal-free reagents

535140

Protease Inhibitor Cocktail Set IV

AEBSF, HCl; E-64 Protease Inhibitor;Pepstatin A; o-Phenanthroline

Fungal and yeast cell extracts 539136

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

539137

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 animal-free reagents

535141

Protease Inhibitor Cocktail Set VI

AEBSF, HCl; Bestatin; E-64 Protease Inhibitor; Leupeptin, Hemisulfate; o-Phenanthroline; Pepstatin A

Plant cell extracts 539133

Protease Inhibitor Cocktail Set VII

AEBSF, HCl; Bestatin; E-64 Protease Inhibitor; Pepstatin A; Phosphoramidon, Disodium Salt

Proteins containing His tag sequences 539138

Protease Inhibitor Cocktail Set VIII

ALLN; Cathepsin Inhibitor I; E-64 Protease Inhibitor

Broad range cysteine protease inhibition 539129

Serine Protease Inhibitor Cocktail Set I

AEBSF, HCl; Aprotinin, Bovine Lung; Elastatinal, GGACK

Broad range serine protease inhibition 565000

Protease Inhibitor Set AEBSF, HCl; E-64; EST; Leupeptin, Hemisulfate ; Pepstatin A; TLCK, HCl; TPCK

For screening for suitable protease inhibitors 539128

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Catalogue No. 539134Form: Liquid

More efficient than competitor product:

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 Cocktail Set III, EDTA-Free

Featured Protease Inhibitor Cocktail

Protease Inhibitor Molecular Weight (MW) Concentration in Vial Target Proteases

AEBSF, hydrochloride 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 Cysteine proteases

Leupeptin, hemisulfate 475.6 2 mM Cysteine proteases and trypsin-like proteases

Pepstatin A 685.9 1 mM Aspartic proteases

Volume of cell extractRequired amount of EMD Millipore Protease

Inhibitor Cocktail Set IIIRequired amount of Supplier R Protease

Inhibitor Cocktail

500 mL 1 mL 10 tablets

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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:

• Stable — our cocktails offer a greater degree of stability compared to tablets dissolved in medium (see Figure 1)

• Efficient — the liquid cocktail format allows immediate exposure of inhibitors to proteases present in samples

• Flexible, minimizing waste — you only need to add the amount of cocktail needed for your specific protocol

• Convenient, easy to use — working reagents are dissolved and ready to use

• Consistent — 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.

Figure 1. Stability of Protease Inhibitor Dilutions in BugBuster® Lysis ReagentProtease inhibitors were diluted to the prescribed working concentration (Competitor or Calbiochem® Cocktail VII, Catalogue No. 539138). The ability of the inhibitors to inhibit the proteolytic activity of PRONASE® (Catalogue No. 537088) 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® (Catalogue No. 537088), 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.

Calbiochem® Protease Inhibitors offer greater efficiency and stability

40000

70000

60000

50000

30000

20000

10000

0No Inhibitors A B

day 0 A = Competitor, 8 °C

day 1 B = Calbiochem® Cocktail VII, 8 °C

day 3 C = Competitor, room temp.

day 5 D = Calbiochem® Cocktail VII, room temp

C D

Prot

eoly

tic A

ctiv

ity

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Description Catalogue No.

Protease Inhibitor Cocktail Set I 539131

Protease Inhibitor Cocktail Set I, Animal-Free 535142

Protease Inhibitor Cocktail Set II 539132

Protease Inhibitor Cocktail Set III, EDTA-Free 539134

Protease Inhibitor Cocktail Set III, Animal-Free 535140

Protease Inhibitor Cocktail Set IV 539136

Protease Inhibitor Cocktail Set V, EDTA-Free 539137

Protease Inhibitor Cocktail Set V, Animal-Free 535141

Protease Inhibitor Cocktail Set VI 539133

Protease Inhibitor Cocktail Set VII 539138

Protease Inhibitor Cocktail Set VIII 539129

Serine Protease Inhibitor Cocktail Set I 565000

Protease Inhibitor Set 539128

Protease Arrest™ Reagent 539124

Available from www.emdbiosciences.com and www.millipore.com

Key Products

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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.

Phosphatase Inhibitor Cocktails

For our current pricing or to download comprehensive information on these products, visit www.emdbiosciences.com/phosphataseinhibitors

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.

Application-Specific Phosphatase Inhibitor Cocktails

Product Components Application Catalogue No.

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

524624

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)

524625

Phosphatase Inhibitor Cocktail Set III

Sodium Fluoride; Sodium Orthovanadate; Sodium Pyrophosphate, decahydrate; b-Glycerophosphate

Protection against Ser/Thr phosphatases and protein tyrosine phosphatases (PTPs)

524627

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

524628

PhosphoSafe™ Extraction Reagent

Protection against Ser/Thr phosphatases and Protein Tyrosine Phosphatases (PTPs)

71296

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Catalogue No. 524625Form: 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 Cocktail Set II

Featured Phosphatase Inhibitor Cocktail

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 extractRequired amount of EMD Millipore

Phosphatase Inhibitor Cocktail Set IIIRequired amount of Supplier R Phosphatase Inhibitor Cocktail

500 mL 1 set (5 x 1 mL) 50 tablets

Description Catalogue No.

Phosphatase Inhibitor Cocktail Set I 524624

Phosphatase Inhibitor Cocktail Set II 524625

Phosphatase Inhibitor Cocktail Set III 524627

Phosphatase Inhibitor Cocktail Set III, Lyophilized 524631

Phosphatase Inhibitor Cocktail Set IV 524628

Phosphatase Inhibitor Cocktail Set V 524629

Phosphatase Inhibitor Cocktail Set V, 50X, Lyophilized 524632

PhosphoSafe™ Extraction Reagent 71296

Available from www.emdbiosciences.com and www.millipore.com

Key Products

10

Description Catalogue No.

BugBuster® Protein Extraction Reagent 70584

BugBuster® Master Mix Extraction reagent 71456

BugBuster® Plus Benzonase Nuclease 70750

BugBuster® Plus Lysonase™ Kit 71370

BugBuster® HT Protein Extraction Reagent 70922

BugBuster® 10X Protein Extraction Reagent 70921

BugBuster®(primary amine-free) Extraction Reagent 70923

PopCulture® Reagent 71092

Insect PopCulture® Reagent 71187

YeastBuster™ Protein Extraction Reagent 71186

CytoBuster™ Protein Extraction Reagent 71009

NucBuster™ Protein extraction Reagent 71183

ProteoExtract® Subcellular Protein Extraction Kit 539790

ProteoExtract® Native Membrane Protein Extraction Kit 444810

ProteoExtract® Transmembrane Protein Extraction Kit 71772

ProteoExtract® Complete Mammalian Protein Extraction Kit 539779

ProteoExtract® Cytosol/Mitochondria Fractionation Kit QIA88

ProteoExtract® Tissue Dissociation Kit 539720

ProteoExtract® All-in-One Trypsin Digestion Kit 650212

ProteoExtract® Phosphopeptide Enrichment TiO2 Kit 539722

ProteoExtract® Phosphopeptide Enrichment SCIMAC Kit 539723

ProteoExtract® Glycopeptide Enrichment Kit 72103

ProteoExtract® Native Cytoskeleton Enrichment Kit 17-10210 n

ProteoExtract® Cytoskeleton Enrichment & Isolation Kit 17-10195 n

Available from www.emdbiosciences.com

Related Products

n Available from www.millipore.com

Amicon® Ultra-0.5

Amicon® Ultra-2

Amicon® Ultra-4

Amicon® Ultra-15

11

Description Catalogue No.

PureProteome™ Nickel Magnetic Beads LSKMAGH10

PureProteome™ Protein A Magnetic Beads LSKMAGA10

PureProteome™ Protein G Magnetic Beads LSKMAGG10

PureProteome™ Albumin Magnetic Beads LSKMAGL10

PureProteome™ Albumin/IgG Depletion Kit LSKMAGD12

PureProteome™ Streptavidin Magnetic Beads LSKMAGT10

PureProteome™ NHS FlexiBind Magnetic Beads LSKMAGN04

PureProteome™ 8 place Magnetic Stand LSKMAGS08

PureProteome™ 2 place Magnetic Stand LSKMAGS15

PureProteome™ 0.3 µm Carboxylic Acid FlexiBind Magnetic Beads, 2 mL LSKMAG03CBX02

PureProteome™ 1.0 µm Carboxylic Acid FlexiBind Magnetic Beads, 2 mL LSKMAG1CBX02

PureProteome™ 2.5 µm Carboxylic Acid FlexiBind Magnetic Beads, 2 mL LSKMAG25CBX02

Protein Purification, Concentration and Buffer Exchange

For extremely fast and easy protein purification, trust PureProteome™ magnetic bead systems, which feature low non-

specific binding and minimal sample loss. Conventional purification methods require centrifugation to pellet, followed by

careful aspiration to avoid losing sample. Magnetic beads are isolated using a magnetic stand, enabling total removal of

buffers and complete recovery of beads with no sample loss.

Amicon® Ultra centrifugal filters provide fast sample processing and promote high sample recoveries, even in dilute

samples, through ultrafiltration. The vertical membrane design, true dead stop, reverse spin recovery option and broad

chemical compatibility of Amicon® Ultra centrifugal filters give you the fastest, most efficient concentration for sensitive

downstream applications.

Amicon® Ultra filters are available in various sizes and molecular weight cut offs (MWCO) to match your starting volume,

molecular weight of protein or nucleic acid being concentrated, final volume and concentration factor.

Use the online Amicon® Ultra selector tool to choose the perfect filter and view protocols:

www.millipore.com/AmiconSelect

PureProteome™ Magnetic Beads for Protein Purification

Amicon® Ultra Centrifugal Filters

Available from www.millipore.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. PB3338EN00 LS-SBU-11-05727 Printed in the USA 01/2012 © 2012 EMD Millipore Corporation, Billerica, MA, USA. All rights reserved.

www.emdmillipore.com

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