Broad Spectrum Protease Inhibitor Cocktail (100x)

List of Components

Overview

Notes

BioChemical Information

Assay Principle

Broad Spectrum Protease Inhibitor Cocktail (100X) is a Western blot related ready-to-use concentrated stock solution reagent containing a blend of seven protease inhibitors that is to be added to cell lysis buffer to protect the integrity and functionality of native cellular proteins against degradation by multiple classes of endogenous proteases during protein extraction and sample preparation procedures. The product is supplied as a 100X concentrated stock solution in a liquid format for improved accuracy, solubility, and ease of use in comparison to traditional tablets.

Properties

Background

Crude cell extracts contain a number of endogenous enzymes, such as proteases and phosphatases, which are capable of digesting the proteins present in the extract. An optimized method to improve the yield of intact and functional native proteins is to add inhibitors of these enzymes known to be present in the source material. This broad spectrum protease inhibitor cocktail is a complex of various protease inhibitors, which has been tested for inhibiting proteases and esterase broadly.
Protease inhibitors are critical reagents for the preservation of protein integrity during purification and analysis procedures by knocking out specific proteases to avoid peptide bond hydrolysis and subsequent protein destruction. They are biological or chemical compounds that function by reversibly or irreversibly binding to the protease. Most known proteases belong to one of four evolutionarily distinct enzyme families based on the functional groups involved in the peptide bond cleavage. No single chemical is effective for all types of proteases. A mixture or "cocktail" of several different inhibitor compounds is commonly used that consistently inhibit a multitude of protease classes to ensure that protein extracts do not degrade before analysis for target proteins of interest. Not using protease inhibitors leads to the loss of a large number of valuable proteins in a lysate sample, adversely affecting downstream applications by biologically meaningless representation of protein activities and gaining false negative immunostaining results for targets of interest.

Usage and Handling

Notes:

• Equilibrate the bottle to room temperature before use.

• Vortex the bottle before use to ensure a homogeneous suspension

• This protease inhibitor cocktail is supplied at a 100X concentration in DMSO and is generally effective when used at a 1X final concentration; however, if a sample contains particularly high levels of proteases, the effective cocktail concentration might require optimization.

1. Add broad spectrum protease inhibitor cocktail in lysis buffer at a ratio of 1:100 and mix well.

2. Optional: For inhibition of metalloproteases, add 0.1M EDTA solution in lysis buffer at a ratio of 1:100 and mix well.

3. Add the solution in to cell or tissue samples for protein extraction.
Lysis buffer containing protease inhibitor cocktail should be freshly prepared before use.

Validation Images & Assay Conditions

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Protease activity for extracted protein from mouse tissue while using different protease inhibitor cocktails