Western blot analysis of extracts from NIH/3T3 cells, prepared in lysis buffer in the absence of protease inhibitors (left) or with Protease Inhibitor Cocktail (100X) #5871 added (right), and incubated at 37ºC for the indicated time points, using p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695. In the absence of protease inhibitors, p44/42 MAPK signal fades within 6 hrs. after harvest, indicating protein degradation. In the presence of the protease inhibitor cocktail, the p44/42 MAPK degradation is slowed significantly and signal is still present at 48 hrs. following harvest.
1. Briefly vortex the Protease 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 Inhibitor Cocktail (100X) is composed of a proprietary mix of AEBSF, Aprotinin, Bestatin, E64, Leupeptin, and Pepstatin A to promote broad spectrum protection against endogenous proteases. 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 5 mM.
Store the undiluted 100X cocktail at 4ºC. Do not freeze.
When diluted in lysis buffer to a final concentration of 1X the Protease Inhibitor Cocktail prevents protein degradation by endogenous proteases present in whole cell extract. The 100X
Protease Inhibitor Cocktail is a clear, colorless liquid.
In order to study specific target proteins of interest protease-mediated degradation during the generation of protein lysates is to be avoided. A loss of normal cellular control occurs during cell lysis, and endogenous proteases within the cell extract are free to degrade proteins in an uncontrolled manner. 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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