1. Briefly vortex the Protease/Phosphatase Inhibitor Cocktail (100X) before use.
2. Just prior to lysing cells, dilute the cocktail 1:100 in desired lysis buffer to obtain a 1X working concentration.
The Protease/Phosphatase Inhibitor Cocktail (100X) is composed of a proprietary mix of Aprotinin, Bestatin, E64, and Leupeptin to promote broad spectrum protection against endogenous proteases and sodium fluoride, sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate to promote broad spectrum protection against endogenous serine/threonine and tyrosine phosphatases. The cocktail does not contain EDTA (a metalloprotease inhibitor) which can be incompatible with some downstream applications (i.e. protein assays, 2D electrophoresis, etc.). If EDTA is desired as a protease inhibitor it can be added to the cell lysis buffer at a final working concentration of 5mM.
Dynamic protein phosphorylation is a key cellular signaling mechanism by which a broad spectrum of cellular processes is regulated. In order to study the phosphorylation status of specific target proteins the phosphorylated residue of interest must remain intact. When cells are lysed to make whole cell extracts, a loss of normal cellular signaling regulation occurs, and phosphatases within the cell extract are free to dephosphorylate proteins in an uncontrolled manner. The addition of phosphatase inhibitors to the cell lysis buffer aids in the preservation of phosphorylated residues at the time of cell disruption.
This same loss of normal cellular control when generating whole cell extracts also leads to uncontrolled degradation of proteins by endogenous proteases. The addition of protease inhibitors to the cell lysis buffer aids in the preservation of target proteins in the cell extract.
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