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Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide for the Use of Specialized Protease Inhibitor Cocktails 3 Protease Inhibitor Cocktail Sets 3 Frequently Asked Questions 7 Properties of Individual Protease Inhibitors 8 Selection Guide for the use of Specialized Phosphatase Inhibitor Cocktails 9 Phosphatase Inhibitor Cocktail Sets 9 Selected Protein Phosphatase Inhibitors 11

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Page 1: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Protease and Phosphatase Inhibitor CocktailsApplication & Product Selection Guide

In This Guide:

Selection Guide for the Use ofSpecialized Protease Inhibitor Cocktails . . 3

Protease Inhibitor Cocktail Sets . . . . . . . 3

Frequently Asked Questions . . . . . . . . . 7

Properties of IndividualProtease Inhibitors . . . . . . . . . . . . . . 8

Selection Guide for the use of Specialized Phosphatase Inhibitor Cocktails . . . . . . . 9

Phosphatase Inhibitor Cocktail Sets . . . . 9

Selected Protein Phosphatase Inhibitors . . 11

Page 2: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Protease Inhibitor Cocktails and Inhibitor Sets Protease inhibitor cocktails and highly specific inhibitors to proteases are important tools for scientists engaged in a variety of proteomic studies including protein expression and 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.

During protein expression and isolation, endogenous proteases rapidly begin to degrade protein samples following cell lysis or tissue disaggregation. The resulting proteolysis can drastically reduce the quality and quantity of protein samples required for characterization and analysis. In order to overcome this problem, complex protein solutions are treated with a mixture or cocktail of protease inhibitors, which help preserve the activity and nature of proteins. By utilizing a specific combination of protease inhibitors, preparative protein samples may be protected from the most common proteases including serine proteases, metalloproteases, cysteine proteases, aminopeptidases, and aspartic proteases.

Using a combination of synthetic, recombinant, and naturally occurring protease inhibitors, we have developed a panel of 12 application specific protease inhibitor cocktails. These cocktails are formulated using reagents of the highest quality and biological activity. A convenient summary of the use of these cocktails is outlined on page 3.

Key features:• Convenient: Protocol flexible, Ready-To-Use formulations• Consistent: High quality ensures reproducibility and excellent inhibition over a wide range of

protease classes• Flexible: A comprehensive selection of specific cocktail formulations designed to inhibit

proteolytic activity from most tissue or cell type extracts, including mammalian, bacterial, yeast, fungal, and plant cells

• Application Specific Formulations:- Available without EDTA for purification schemes involving metal ion chelating

chromatography or analysis using 2-D electophoresis– Option of new protease inhibitor cocktail formulations using recombinant aprotinin for

applications that require the use of animal-free reagents

Calbiochem • Protease and Phosphatase Inhibitor Cocktails� For more information or to place an order, contact your local office (see back cover) .

Page 3: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Protease Inhibitor Cocktail Set IA cocktail containing five protease inhibitors that will inhibit a broad range of proteases. Reconstitute each vial with 1 ml H2O to obtain a 100X stock solution. When diluted to 1X stock solution, contains the concentrations of inhibitors shown in the table on the right.

Cat. No. 539131 1 vial 10 vials

Protease Inhibitor M.W. 1X Concentration Target Proteases

AEBSF, Hydrochloride �39 .5 500 μM Serine Proteases

Aprotinin, Bovine Lung 651� 150 nM Serine Proteases and Esterases

E-64 Protease Inhibitor 357 .4 1 μM Cysteine Proteases

EDTA, Disodium 37� .� 500 μM Metalloproteases

Leupeptin, Hemisulfate 475 .6 1 μM Cysteine Proteases and Trypsin-like Proteases

N Protease Inhibitor Cocktail Set I, Animal-FreeThis animal-free formulation includes the same five pro-tease inhibitors as the original version (Cat. No. 539131), but substitutes Aprotinin, Bovine Lung with a recombinant Aprotinin . Reconstitute each vial with 1 ml H2O to obtain a 100X stock solution. Dilute to 1X before use.

Cat. No. 535142 1 ml 1 set (10 x 1 ml)

Protease Inhibitor M.W. 1X Concentration Target Proteases

AEBSF, Hydrochloride �39 .5 500 mM Serine Proteases

Aprotinin, Recombinant 651� 150 nM Serine Proteases and Esterases

E-64 Protease Inhibitor 357 .4 1 mM Cysteine Proteases

EDTA, Disodium 37� .� 500 mM Metalloproteases

Leupeptin, Hemisulfate 475 .6 1 mM Cysteine Proteases and Trypsin-like Proteases

Selection Guide for the Use of Specialized Protease Inhibitor Cocktails Product Cat. No. Recommended Application

Protease Inhibitor Cocktail Set I 539131 General use

N Protease Inhibitor Cocktail Set I, Animal-Free 535142 General use and for applications which require the use of animal-free reagents

Protease Inhibitor Cocktail Set II 539132 Bacterial cell extracts (except those intended for metal chelation chromatography)

Protease Inhibitor Cocktail Set III, EDTA-Free 539134 Mammalian cells and tissue extracts purified using metal chelation chromatography; samples to be analyzed by �-D gel electrophoresis

N Protease Inhibitor Cocktail Set III, Animal-Free 535140 Mammalian cells and tissue extracts and for applications which require the use of animal-free reagents

Protease Inhibitor Cocktail Set IV 539136 Fungal and yeast cell extracts

Protease Inhibitor Cocktail Set V, EDTA Free 539137 Mammalian cells and tissue extracts purified using metal chelation chromatography; samples to be analyzed by �-D gel electrophoresis

N Protease Inhibitor Cocktail Set V, Animal-Free 535141 Mammalian cells and tissue extracts and for applications which require the use of animal-free reagents

Protease Inhibitor Cocktail Set VI 539133 Plant cell extracts

Protease Inhibitor Cocktail Set VII 539138 Proteins containing His•Tag® sequences

Protease Inhibitor Cocktail Set VIII 539129 Broad range cysteine protease inhibition

Serine Protease Inhibitor Cocktail Set I 565000 Broad range serine protease inhibition

Calbiochem • Protease and Phosphatase Inhibitor Cocktails 3customer .service@merckbio .comtechnical .service@merckbio .comVisit our website www .merckbio .com

Page 4: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Protease Inhibitor Cocktail Set III, EDTA-FreeThis cocktail is recommended for use with mammalian cells and tissue extracts and for bacterial cell extracts being used for metal 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. Each vial contains the concen-trations of inhibitors shown in the table on the right. One ml is sufficient for about 20 g tissue.

Cat. No. 539134 1 ml 1 set (5 x 1 ml)

Protease Inhibitor M.W. Concentration in the Vial

Target Proteases

AEBSF, Hydrochloride �39 .5 100 mM Serine Proteases

Aprotinin, Bovine Lung 651� 80 μM Serine Proteases and Esterases

Bestatin 308 .4 5 mM Aminopeptidase B and Leucine Aminopep-tidase

E-64 Protease Inhibitor 357 .4 1 .5 mM Cysteine Proteases

Leupeptin, Hemisulfate 475 .6 � mM Cysteine Proteases and Trypsin-like Proteases

Pepstatin A 685 .9 1 mM Aspartic Proteases

Protease Inhibitor Cocktail Set IIThis cocktail is recommended for use with bacterial cell extracts (except those intended for metal chelation chroma-tography). Cocktail contains five protease inhibitors with broad specificity for the inhibition of aspartic, cysteine, serine, and metalloproteases as well as aminopeptidases. Reconstitute each vial with 1 ml DMSO and 4 ml H2O to obtain a 5 ml stock solution. When reconstituted each vial will contain the concentrations of inhibitors shown in the table on the right. One set is sufficient for about 20 g cells.Note: 1 set = 1 vial of lyophilized protease inhibitor cocktail and 1 vial DMSO, 1 ml.

Cat. No. 539132 1 set 5 sets

Protease Inhibitor M.W. Concentration in the Vial

Target Proteases

AEBSF, Hydrochloride �39 .5 �0 mM Serine Proteases

Bestatin 308 .4 1 .7 mM Aminopeptidase B and Leucine Aminopep-tidase

E-64 Protease Inhibitor 357 .4 �00 μM Cysteine Proteases

EDTA, Disodium 37� .� 85 mM Metalloproteases

Pepstatin A 685 .9 � mM Aspartic Proteases

N Protease Inhibitor Cocktail Set III, Animal-FreeThis animal-free formulation includes the same six protease inhibitors as the original version (Cat. No. 539134), but substitutes Aprotinin, Bovine Lung with a recombinant Aprotinin. Each vial contains the concentrations of inhibitors shown in the table below. One ml is sufficient for about 20 g tissue.

Cat. No. 535140 1 ml 1 set (5 x 1 ml)

Protease Inhibitor M.W. Concentration in Vial

Target Proteases

AEBSF, Hydrochloride �39 .5 100 mM Serine Proteases

Aprotinin, Recombinant 651� 80 mM Serine Proteases and Esterases

Bestatin 308 .4 5 mM Aminopeptidase B & Leucine Aminopeptidase

E-64 Protease Inhibitor 357 .4 1 .5 mM Cysteine Proteases

Leupeptin, Hemisulfate 475 .6 � mM Cysteine Proteases and Trypsin-like Proteases

Pepstatin A 685 .9 1 mM Aspartic Proteases

Calbiochem • Protease and Phosphatase Inhibitor Cocktails4 For more information or to place an order, contact your local office (see back cover) .

Page 5: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

N Protease Inhibitor Cocktail Set V, Animal-FreeThis animal-free formulation includes the same four protease inhibitors as the original version (Cat. No. 539137), but substitutes Aprotinin, Bovine Lung with a recombinant Aprotinin. Reconstitute each vial with 1 ml H2O to obtain a 100X stock solution. Dilute to 1X before use.

Cat. No. 535141 1 ml 1 set (10 x 1 ml)

Protease Inhibitor Cocktail Set V, EDTA-FreeAn EDTA-free cocktail containing four protease inhibitors for the inhibition of serine and cysteine, but not metalloproteases. Reconstitute each vial with 1 ml H2O to obtain a 100X stock solution. When diluted to 1X stock solution, the set will contain the concentrations of inhibitors shown in the table on the right.

Cat. No. 539137 10 vials

Protease Inhibitor M.W. 1X Concentration Target Proteases

AEBSF, Hydrochloride �39 .5 500 μM Serine Proteases

Aprotinin, Bovine Lung 651� 150 nM Serine Proteases and Esterases

E-64 Protease Inhibitor 357 .4 1 μM Cysteine Proteases

Leupeptin, Hemisulfate 475 .6 1 μM Cysteine Proteases and Trypsin-like Proteases

Protease Inhibitor Cocktail Set IVThis cocktail is recommended for fungal and yeast cell extracts. Cocktail contains four protease inhibitors(in 1 ml DMSO) with broad specificity for the inhibition of aspartic, cysteine, metallo, and serine proteases. Each vial contains the concentrations of inhibitors shown in the table on the right.

Cat. No. 539136 1 ml 1 set

Protease Inhibitor M.W. Concentration in the Vial

Target Proteases

AEBSF, Hydrochloride �39 .5 100 mM Serine Proteases

E-64 Protease Inhibitor 357 .4 1 .5 mM Cysteine Proteases

Pepstatin A 685 .9 � mM Aspartic Proteases

o-Phenanthroline 198 .� 500 mM Metalloproteases

Protease Inhibitor M.W. 1X Concentration Target Proteases

AEBSF, Hydrochloride �39 .5 500 mM Serine Proteases

Aprotinin, Recombinant 651� 150 nM Serine Proteases and Esterases

E-64 Protease Inhibitor 357 .4 1 mM Cysteine Proteases

Leupeptin, Hemisulfate 475 .6 1 mM Cysteine Proteases and Trypsin-like Proteases

Protease Inhibitor Cocktail Set VIThis cocktail is recommended for use with plant cell extracts. Cocktail contains six protease inhibitors (in 1 ml DMSO) with broad specificity for the inhibition of aspartic, cysteine, serine, and metalloproteases as well as aminopep-tidases. Each vial contains the concentrations of inhibitors shown in the table on the right. One ml is recommended for about 30 g of various plant tissues.

Cat. No. 539133 1 ml 1 set (5 x 1 ml)

Protease Inhibitor M.W. Concentration in the Vial

Target Proteases

AEBSF, Hydrochloride �39 .5 �00 mM Serine Proteases

Bestatin 308 .4 10 mM Aminopeptidase B and Leucine Aminopep-tidase

E-64 Protease Inhibitor 357 .4 3 mM Cysteine Proteases

Leupeptin, Hemisulfate 475 .6 � mM Cysteine Proteases and Trypsin-like Proteases

o-Phenanthroline 198 .� 500 mM Metalloproteases

Pepstatin A 685 .9 � mM Aspartic Proteases

Calbiochem • Protease and Phosphatase Inhibitor Cocktails 5customer .service@merckbio .comtechnical .service@merckbio .comVisit our website www .merckbio .com

Page 6: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Protease Inhibitor M.W. Concentration in the Vial

Target Proteases

AEBSF, Hydrochloride �39 .5 100 mM Serine Proteases

Bestatin 308 .4 5 mM Aminopeptidase B and Leucine Aminopep-tidase

E-64 Protease Inhibitor 357 .4 1 .5 mM Cysteine Proteases

Pepstatin A 685 .9 � mM Aspartic Proteases

Phosphoramidon, Disodium

587 .5 �00 μM Metalloendopeptidases

Protease Inhibitor Cocktail Set VIIThis cocktail is recommended for purification of proteins containing His•Tag® sequences. Cocktail contains five protease inhibitors (in 1 ml DMSO) with broad specificity for the inhibition of cysteine, serine, aspartic, and thermolysin-like proteases and aminopeptidases. Each vial contains the concentrations of inhibitors shown in the table on the right. One ml is sufficient for about 10 g cells.

Cat. No. 539138 1 ml 1 set (5 x 1 ml)

Protease Inhibitor M.W. Concentration in the Vial

Target Proteasess

ALLN 383 .5 1 .56 mM Calpain I/II, Cathepsin B, Cathepsin L .,Cysteine Proteases

Cathepsin Inhibitor I 475 .5 500 μM Cathepsin B, Cathepsin L, Cathepsin S, Papain

E-64 Protease Inhibitor 357 .4 1 .5 mM Cysteine Protease

Protease Inhibitor Cocktail Set VIIIA DMSO solution of three protease inhibitors with selective specificity for the inhibition of cysteine proteases, includ-ing calpains, cathepsins, and papain. Each vial contains the concentrations of inhibitors shown in the table on the right.

Cat. No. 539129 1 ml 1 set (5 x 1 ml)

Serine Protease Inhibitor Cocktail Set I A cocktail of four protease inhibitors that is useful for the inhibition of a broad range of serine proteases. Reconstitute each vial with 1 ml H2O to obtain a 100X stock solution. When diluted to 1X stock solution, the set will contain the concentrations of inhibitors shown in the table on the right.

Cat. No. 565000 1 vial 5 vials

Protease Inhibitor 1X stockconcentration

Target Proteases

AEBSF, HCl 500 μM Serine Proteases

Aprotinin, Bovine Lung 4�0 nM Serine Proteases and Esterases

Elastatinal �0 μM An inhibitor of elastase-like serine protease produced by actinomy-cetes .

GGACK 1 μM An irreversible inhibitor of Urokinase and Factor Xa .

Protease Inhibitor M.W. Quantity supplied Target Proteases

AEBSF, HCl �39 .5 50 mg Serine Proteases

E-64 Protease Inhibitor 357 .4 1 mg Cysteine Proteases

EST 34� .4 1 mg Calpains

Leupeptin, Hemisulfate 475 .6 5 mg Cysteine Proteases and Trypsin-like Proteases

Pepstatin A 685 .9 5 mg Aspartic Proteases

TLCK, HCl 369 .3 50 mg Trypsin-like Serine Proteases

TPCK 351 .5 �50 mg Chymotrypsin-like Proteases

Protease Inhibitor SetThis unique set, consisting of seven different inhibitors, allows you to perform easy, economical screening for a suitable inhibitor for your application. Calbiochem® now offers several protease inhibitors with a wide range of specificities packaged in one convenient set.

Cat. No. 539128 1 set

Calbiochem • Protease and Phosphatase Inhibitor Cocktails6 For more information or to place an order, contact your local office (see back cover) .

Page 7: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

0

10000

20000

30000

40000

50000

60000

70000

Prot

eoly

tic A

ctiv

ity

day 0

day 1

day 3

day 5

A = Competitor, 8°CB = Calbiochem® Cocktail VII, 8°C C = Competitor, room temp.D = Calbiochem® Cocktail VII, room temp.

No Inhibitors A B C D

Protease Arrest™ ReagentAn optimized concentration of various reversible and irreversible inhibitors to inhibit serine, cysteine, and calpain proteases. Suitable for the protection of proteins purified from animal tissues, plant tissues, yeast, and bacteria. Protease Arrest Reagent is provided as a 50x solution that when diluted in extraction buffer at pH 7.0–8.0 inhibits 95–98% of protease activity. EDTA is also provided separately to inhibit metalloproteases.

Cat. No. 539124 1 set

Frequently Asked Questions

Stability of Protease Inhibitor Dilutions in BugBuster®Protease inhibitors were diluted to the prescribed working concentration (Competitor; Calbiochem® Cocktail VII, Cat. No. 539138). The ability of the inhibitors to inhibit the proteolytic activity of PRONASE (Cat. 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 (FTC-casein). 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. 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 shows that the efficiency of the cocktail remained higher than the competitor’s tablet in this study.

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 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:

• Efficient – the liquid cocktail format allows immediate exposure of inhibitors to proteases present in samples.• Flexible and economical - add only protocol-specific cocktail volumes as necessary • Easy-to-use - working reagents are dissolved and ready to use • Consistent - the same quantity of inhibitor is always dispensed • Stable - our cocktails offer a greater degree of stability compared to tablets dissolved in medium.

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 with use on liquid handlers and are stable as shown in the data below.

More online... www.calbiochem.com/inhibitorcocktails

Calbiochem • Protease and Phosphatase Inhibitor Cocktails 7customer .service@merckbio .comtechnical .service@merckbio .comVisit our website www .merckbio .com

Page 8: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Properties of Individual Protease Inhibitors Protease Inhibitor Cat. No. Target Protease Class and Mechanism of Action Solubility Suggested Concentration Size

AEBSF, Hydrochloride 101500 Water soluble, non-toxic alternative to PMSF . Irreversible inhibitor of serine proteases . Reacts covalently with a component of the active site . Inhibits chymotrypsin, kali-krein, plasmin, trypsin and related thrombolytic enzymes .

H�O 0 .1-1 mM 50 mg100 mg500 mg

ALLN 208719 Inhibits plasmin, thrombin, calpain I (Ki = 190 nM), calpain II (Ki = ��0 nM), Cathepsin B (Ki = 150 nM), and cathepsin L (Ki = 600 pM) .

Methanol, Ethanol, DMSO

0 .�-� μM 5 mg�5 mg

Aprotinin, Bovine Lung 616370 A serine protease inhibitor that acts as a competitive and reversible inhibitor of proteolytic and esterolytic activity . In cell cultures, extends the life of cells and prevents proteolytic damage to intact cells .

H�O 0 .6-� .0 μg/ml 10 mg�0 mg

100 mg

Aprotinin, Recombinant 616371 A serine protease inhibitor that acts as a competitive and reversible inhibitor of proteolytic and esterolytic activity . In cell cultures, extends the life of cells and prevents proteolytic damage to intact cells .

H�O 0 .6-� .0 μg/ml 1 mg5 mg

�5 mg

Bestatin 200484 Binds to cell surfaces and inhibits cell surface aminopeptidases, notably aminopeptidase B and leucine aminopeptidase . Activates macrophages andT lymphocytes . Has antitumor properties .

Methanol 1-10 μM 10 mg

Cathepsin Inhibitor I 219415 Selectively inhibits cathepsin B (k�/Ki = 6 .9 x 103 M-1 sec-1), cathepsin L (k�/Ki = 3 .1 x 105 M-1 sec-1), cathepsin S (k�/Ki = 6 .6 x 104 M-1 sec-1), and papain (k�/Ki = 1 .8 x 103 M-1 sec-1) .

DMSO, Ethanol

100-�00 μM 1 mg

E-64 Protease Inhibitor 324890 An irreversible cysteine protease inhibitor that has no action on cysteine residues in other proteins . Specific active site titrant .

H�0, DMSO 1-10 mM 1 mg5 mg

�5 mg

EDTA, Disodium 324503 A reversible metalloprotease inhibitor . A chelator that may interfere with other metal ion-dependant biological processes .

H�O 1-10 mM 100 g1 Kg

Elastatinal 324691 A competitive inhibitor of elastase (Ki = �40 nM) . H�O, DMSO, Ethanol

0 .5-� μg/ml 5 mg

EST 330005 A membrane permeable calpain inhibitor . Ethanol �0-50 μg/ml 1 mg

GGACK 347436 An irreversible inhibitor of Urokinase (IC50 < 1 μM) . H�O 1-10 mM 5 mg

Leupeptin, Hemisulfate 108975 A reversible inhibitor of trypsin-like proteases and cysteine proteases, including endoproteinase Lys-C, papain, cathepsin B, trypsin, kallikrein, and thrombin .

H�O 10-100 μM 5 mg10 mg�5 mg50 mg

100 mg

Pepstatin A 516481 A reversible inhibitor of aspartic protease . Inhibits cathep-sin D, cathepsin G, pepsin, and renin .

DMSO, Methanol

~ 1 μM 5 mg�5 mg

100 mg

o-Phenanthroline 516705 A metalloprotease inhibitor . DMSO,Ethanol, H�O

500 mg

Phosphoramidon, Disodium Salt

525276 A highly specific inhibitor of thermolysin . Also inhibits the conversion of big endothelin-1 to endothelin-1 .

H�O, DMSO, Methanol

1-10 μM 5 mg

TLCK, HCl 616382 An irreversible inhibitor of trypsin-like serine proteases . Inactivates trypsin, specifically and irreversible . Does not have any significant inhibitory effect on chymotrypsin .

H�O 10-100 μM 50 mg�50 mg

TPCK 616387 An irreversible inhibitor of chymotrypsin . Useful for inhib-iting chymotrypsin activity in trypsin preparations .

Ethanol, Methanol

10-100 μM �50 mg1 g

N

More online... www.calbiochem.com/proteaseinhibitors

Calbiochem • Protease and Phosphatase Inhibitor Cocktails8 For more information or to place an order, contact your local office (see back cover) .

Page 9: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Phosphatase Inhibitor CocktailsPhosphorylation and dephosphorylation of proteins mediate signal transduction events that control a multitude of cellular processes such as cell division, cell proliferation, apoptosis, etc. 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, that restore the protein to its original dephosphorylated state. During the extraction of phosphorylated proteins from cell and tissue lysates, it is highly critical that the phosphorylation state of the proteins be preserved. EMD Biosciences offers four different Phosphatase Inhibitor cocktails and a PhosphoSafe™ Extraction Reagent that help protect phosphoproteins from different families of phosphatases as shown in the selection guide below.

Phosphatase Inhibitor Cocktail Set IA cocktail of three inhibitors that will inhibit alkaline phosphatases as well as serine/threonine protein phosphatases such as PP1 and PP2A. Each vial contains 1 ml of phosphatase inhibitor cocktail solubilized in DMSO. The concentrations of the individual inhibitors are shown in the table on the right. Note: 1 set = 5 x 1 ml. Not available for sale outside of the United States.

Cat. No. 524624 1 set

Phosphatase Inhibitor Cocktail Set IIA 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 on the right. Note: 1 set = 5 x 1 ml.

Cat. No. 524625 1 set

Product Cat . No . Recommended Application

Phosphatase Inhibitor Cocktail Set I 524624 Protection against alkaline phosphatases and Ser/Thr phosphatases such as PP1 and PP�A

Phosphatase Inhibitor Cocktail Set II 524625 Protection against acid and alkaline phosphatases and Protein Tyrosine Phosphatases (PTPs)

Phosphatase Inhibitor Cocktail Set III 524627 Protection against Ser/Thr phosphatases and Protein Tyrosine Phosphatases (PTPs)

Phosphatase Inhibitor Cocktail Set IV 524628 Protection against alkaline phosphatases and Ser/Thr phosphatases such as PP1 and PP�A

PhosphoSafe™ Extraction Reagent 71296 Protection against Ser/Thr phosphatases and Protein Tyrosine Phosphatases (PTPs)

Phosphatase Inhibitor M.W. Concentration in Vial

Target Phosphatases

(-)-p-Bromotetramisole oxalate

373 .� � .5 mM Alkaline phosphatases

Cantharidin 196 .� 500 mM PP1 and PP�A

Microcystin LR, Microcystis aeruginosa

995 .� 500 nM PP1 and PP�A

Phosphatase Inhibitor M.W. Concentration in Vial

Target Phosphatases

Imidazole 68 .1 �00 mM Alkaline phosphatase

Sodium Fluoride 4� .0 100 mM Acid phosphatase

Sodium Molybdate �05 .9 115 mM Acid phosphatase

Sodium Orthovanadate 183 .9 100 mM Protein tyrosine phosphatase, Alkaline Phosphatase

Sodium Tartrate, Dihydrate

�30 .1 400 mM Acid phosphatase

Selection Guide for the use of Specialized Phosphatase Inhibitor Cocktails

Calbiochem • Protease and Phosphatase Inhibitor Cocktails 9customer .service@merckbio .comtechnical .service@merckbio .comVisit our website www .merckbio .com

Page 10: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Request your FREE copy today! www.calbiochem.com/inhibitorsourcebook

Includes a comprehensive range ofinhibitors, plus technical articles and tips.

2nd EditionInhibitor SourceBook™

More than1,200 Inhibitors!

N Phosphatase Inhibitor Cocktail Set IIIA cocktail of four phosphatase inhibitors for broad-spectrum inhibition of both serine/threonine and protein tyrosine phosphatases. Available as a 1 ml vial or as a set of five 1 ml vials. Each vial contains 1 ml of aqueous solution of the phosphatase inhibitor cocktail. The concentrations of the individual inhibitors are shown in the table on the right.

Cat. No. 524627 1 ml 1 set

N Phosphatase Inhibitor Cocktail Set IVA cocktail of three phosphatase inhibitors for the inhibition of both serine/threonine and alkaline phosphatases. Available as a 1 ml vial or as a set of five 1 ml vials. Each vial contains a 1 ml solution of the phosphatase inhibitor cocktail. The concentrations of the individual inhibitors are shown in the table on the right.

Cat. No. 524628 1 ml 1 set

Phosphatase Inhibitor M.W. Concentration in Vial

Target Phosphatases

Sodium Fluoride 4� .0 50 mM Acid phosphatases

Sodium Orthovanadate 183 .9 1 mM Protein tyrosine phosphatase, Alkaline Phosphatase

Sodium Pyrophosphate, decahydrate

416 .1 10 mM PP1 and PP�A

b-Glycerophosphate 306 .1 10 mM Ser/Thr phosphatases

Phosphatase Inhibitor M.W. Concentration in Vial

Target Phosphatases

(-)-p-Bromotetramisole oxalate

373 .� � .5 mM Alkaline phosphatases

Cantharidin 196 .� 500 mM PP1 and PP�A

Calyculin A, Discodermia calyx

1009 .� 1 mM PP1 and PP�A

PhosphoSafe™ Extraction Reagent PhosphoSafe™ Extraction Reagent efficiently extracts cytosolic proteins from mammalian and insect cells while preserving their phosphorylation state. This reagent contains the same formulation as CytoBuster™ Protein Extraction Reagent (Trim 2001), but also includes four phosphatase inhibitors: sodium fluoride, sodium vanadate,b-glycerophosphate, and sodium pyrophosphate. PhosphoSafe™ Reagent is compatible with kinase assays, protein interaction analysis, and other applications.

Cat. No. 71296 25 ml 125 ml

Calbiochem • Protease and Phosphatase Inhibitor Cocktails10 For more information or to place an order, contact your local office (see back cover) .

Prices and availability are subject to change . ©Copyright �006 Merck Biosciences, an affiliate of Merck KGaA, Darmstadt, Germany . All rights reserved . Each product is sold with a limited warranty which is provided with each purchase . Each product is intended to be used for research purposes only . It is not to be used for drug or diagnostic purposes nor is it intended for human use . Merck Biosciences products may not be resold, modified for resale, or used to manufacture commercial products without written approval of Merck Biosciences .

Calbiochem®, BugBuster®, and His•Tag® and Pronase® are registered trademarks of EMD Biosciences, Inc . in the United States and in certain other jurisdictions . CytoBuster™, InSolution™, PhosphoSafe™, and Sourcebook™ are trademarks of EMD Biosciences, Inc . Protease Arrest™ is a trademark of Geno Technology, Inc .

Page 11: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

Selected Protein Tyrosine Phosphatase Inhibitors

Product Cat . No . Comments Size

bpV(bipy) �03694 Blocks insulin receptor dephsophorylation (Ki = 100 nM) and inhibits PTEN (IC50 = 18 nM) .

5 mg

bpV(HOpic) �03701 A potent inhibitor of PTEN (IC50 = 14 nM) . 5 mg

bpV(phen) �03695 A potent inhibitor of PTEN (IC50 = 38 nM) . 10 mg

bpV(pic) �03705 A potent inhibitor of PTEN (IC50 = 31 nM) . 5 mg

CDC�5 Phosphatase Inhibitor I, BN8�00� �17691 A potent, selective, and irreversible inhibitor of CDC�5 phosphatase family (IC50 in mM = � .4, 3 .9, 6 .3, 5 .4, and 4 .6 for �5A, �5B�, �5B3, �5C, and �5C-cat, respectively) .

5 mg

Dephostatin �63�00 A competitive PTP inhibitor (IC50 = 7 .7 mM) . 1 mg

mpV(pic) 475950 A PTPase inhibitor and an activator of insulin receptor kinase . 10 mg

Phenylarsine Oxide 5�1000 A PTP inhibitor (IC50 = 18 mM) that blocks TNF-a-dependent activation of NF-kB in human myeloid ML-1a cells .

�50 mg

Protein Tyrosine Phosphatase CD45 Inhibitor

540�15 A cell-permeable, potent, selective, competitive, and reversible inhibitor of CD45 (IC50 = �00 nM using pMPP as substrate) .

1 mg

PTP1B Inhibitor 539741 A selective, reversible and non-competitive allosteric inhibitor of PTP1B (IC50 = 4 mM and 8 mM for PTP1B403 and PTP1B�98, respectively) .

5 mg

Sodium Orthovanadate 567540 A broad-spectrum potent inhibitor of PTPases . 5 g

Sodium Stibogluconate 567565 An irreversible PTP inhibitor that blocks SHP-1 activity (99% inhibition at ~11 mM) . At higher concentrations (~110 mM) also inhibits SHP-� and PTP1B .

1 g

Product Cat . No . Comments Size

Calyculin A, Discodermia calyx �08851 A cell-permeable inhibitor of PP�A (IC50 = 0 .5 - 1 .0 nM) and PP1 (IC50 = � nM) . 10 mg

Cantharidic Acid �10150 A potent inhibitor of PP�A (IC50 = 50 nM) . 10 mg

Cypermethrin �39900 A potent inhibitor of PP�B (IC50 = 40 pM) . 10 mg

DARPP-3�, Phospho-, Rat, Recombinant, E. coli

�51756 A potent inhibitor of PP1 (IC50 = 1 nM) . 30 mg

Deltamethrin �53300 A potent inhibitor of PP�B (IC50 = 100 pM) . 10 mg

eIF-�a Inhibitor, Salubrinal 3�4895 Blocks the activity of PP1 and its nonenzymatic cofactor GADD34. 5 mg

Endothall 3�4760 A specific inhibitor of PP�A (IC50 = 90 nM) . �0 mg

Fenvalerate 341380 A Potent inhibitor of PP�B (IC50 = � - 4 nM) . �5 mg

Microcystin-LR Microcystis aeruginosa 475815 A potent inhibitor of PP1 (IC50 = 1 .7 nM) and PP�A (IC50 = 40 pM) . Not available for sale outside of the United States.

500 mg

NIPP-1, His•Tag®, Bovine Thymus, Recombinant, E. coli

48��51 A potent and specific inhibitor of PP1 (Ki = 1 - 10 pM) . Can be used to distinguish the effect of PP1 from PP�A, PP�B, and PP�C .

1 mg

Okadaic Acid, Prorocentrum concavum 495604 A potent inhibitor of PP1 (IC50 = 10 - 15 nM) and PP�A (IC50 = 100 pM) . 10 mg�5 mg

100 mg

Protein Phosphatase �A Inhibitor I1PP�A,

Human Kidney, Recombinant, E. coli53955� A potent inhibitor of all forms of PP�A (IC50 = ~100 pM) . �50 ng

Protein Phosphatase Inhibitor �, Human, Recombinant, E. coli

539638 The regulatory subunit of the cytosolic type 1 PP1 that inhibits the activity of the free catalytic subunit of PP1 (IC50 = 1 nM) .

100 mg

InSolution™ Tautomycetin,S. griseochromogenes

580550 A selective PP1 inhibitor with ~38-fold greater potency compared to PP�A (IC50 = 1 .6 nM for PP1 and 6� nM for PP�A) .

50 mg

Tautomycin, Streptomyces spiroverticillatus

580551 Inhibits PP1 (IC50 = 1 nM), PP�A (IC50 = 10 nM), and smooth muscle endogenous phosphatase (IC50 = 6 nM) .

50 mg

Selected Serine/Threonine Phosphatase Inhibitors

Selected Protein Phosphatase Inhibitors

More online... www.calbiochem.com/phosphataseinhibitors

Calbiochem • Protease and Phosphatase Inhibitor Cocktails 11customer .service@merckbio .comtechnical .service@merckbio .comVisit our website www .merckbio .com

Page 12: Protease and Phosphatase Inhibitor Cocktails - CTR Scientific · Protease and Phosphatase Inhibitor Cocktails Application & Product Selection Guide In This Guide: Selection Guide

CB0734-�006APLAProtease and Phosphatase Inhibitor Cocktails

Calbiochem, Novabiochem, and Novagen are brands of Merck Biosciences, an affiliate of Merck KGaA, Darmstadt, Germany.

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