Revision 2
Cell Signaling Technology

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3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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Product Information

Product Usage Information

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.

Solutions and Reagents

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.

Storage

Store the undiluted 100X cocktail at 4ºC. Do not freeze. This product is stable for 12 months.

Product Description

When diluted in lysis buffer to a final concentration of 1X the Protease/Phosphatase Inhibitor Cocktail prevents protein degradation and dephosphorylation by endogenous proteases and phosphatases present in the whole cell extract. The 100X cocktail is a clear light yellow to light green liquid.

Background

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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 2
#5872

Protease/Phosphatase Inhibitor Cocktail (100X)

Protease/Phosphatase Inhibitor Cocktail (100X): Image 1 Expand Image
Western blot analysis of extracts from NIH/3T3 cells, prepared in lysis buffer in the absence of protease inhibitors (left) or with Protease/Phosphatase Inhibitor Cocktail (100X) #5872 added (right), and incubated at 37ºC for the indicated time points, using β-Catenin (D10A8) XP® Rabbit mAb #8480. In the absence of protease inhibitors, β-Catenin signal fades within 3 hr after harvest, indicating protein degradation. In the presence of the protease inhibitor cocktail, the β-Catenin degradation is slowed significantly and signal is still present at 20 hr following harvest.
Protease/Phosphatase Inhibitor Cocktail (100X): Image 2 Expand Image
Western blot analysis of extracts from NIH/3T3 cells, serum-starved overnight and treated with hPDGF-BB #8912 (100ng/ml, 5min), prepared in lysis buffer in the absence of phosphatase inhibitors (left) or with Protease/Phosphatase Inhibitor Cocktail (100X) #5872 added (right), and incubated at 37ºC for the indicated time points, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower). In the absence of phophatase inhibitors, phospho-Akt signal drops significantly after time point 0, demonstrating rapid loss of phosphorylation at later time points. In the presence of the phosphatase inhibitor cocktail, the phospho-Akt signal is preserved through all time points monitored.