Western blot analysis of extracts from NIH/3T3 cells, prepared in lysis buffer in the absence of phosphatase inhibitors (left) or with Phosphatase Inhibitor Cocktail (100X) #5870 added (right), and incubated at 37ºC for the indicated time points, using Phospho-PKC (pan) (ΒII Ser660) Antibody #9371. In the absence of phosphatase inhibitors, Phospho-PKC signal fades within 3 hrs. after harvest, indicating protein degradation. In the presence of the phosphatase inhibitor cocktail, the Phospho-PKC degradation is slowed significantly and signal is still present at 24 hrs. following harvest.
Briefly vortex the Phosphatase Inhibitor Cocktail (100X) before use. Then just prior to use:
For Western Blot or Immunoprecipitation Cell Lysis: Dilute the cocktail 1:100 in desired lysis buffer to obtain a 1X working concentration.
For PTMScan(R) Cell Lysis: Dilute the cocktail 1:50 in urea lysis buffer to obtain a 2X working concentration. This higher concentration is required due to the concentrated nature of the lysate.
The Phosphatase Inhibitor Cocktail (100X) is composed of a proprietary mix of sodium fluoride, sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate to promote broad spectrum protection against endogenous serine/threonine and tyrosine phosphatases.
Store the undiluted 100X cocktail at 4ºC. Do not freeze.
This Phosphatase Inhibitor Cocktail is used to prevent dephosphorylation of phosphorylated proteins from active serine/threonine and tyrosine phosphatases present in whole cell extract. The 100X cocktail is a colorless to light yellow liquid.
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.
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