Merck UK: Calbiochem - Protease and Phosphatase Inhibitor Cocktails: FAQ

		Technical Resources

		Technical Information

		Calbiochem Information

		Protease and Phosphatase Inhibitor Cocktails Resource

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Protease Inhibitor Cocktails		Phosphatase Inhibitor Cocktails
•	Overview/Selection Guide	•	Overview/Selection Guide
•	Protease Inhibitor Cocktail Sets	•	Phosphatase Inhibitor Cocktail Sets
•	Properties of Selected Protease Inhibitors	•	Selected Protein Tyrosine Phosphatase Inhibitors
	Frequently Asked Questions	•	Selected Serine/Threonine Phosphatase Inhibitors

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1. What criteria should I use when selecting a protease inhibitor?

2. I currently use protease inhibitor cocktail tablets. Is it easy to switch to ready-to-use liquid cocktails?

3. I work in an automation lab and want to use the cocktails on a liquid handling workstation. Are Calbiochem protease inhibitor cocktails stable?

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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.

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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
•	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

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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.

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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.

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