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SKU AR1121
Pack size80mg (sufficient for 100mL of extract)
Physical StateWhite lyophilized powder
Applications Animal tissue and cell extract with detergent

Overview

Product Name Phosphatase Inhibitor Cocktail III
SKU/Catalog Number AR1121
Physical State White lyophilized powder
Pack Size 80mg (sufficient for 100mL of extract)
Content Proprietary mix of: Sodium fluoride, Sodium orthavanadium, Sodium pyrophosphate, B-phosphoric acid glycerol
Recommended working concentration Reconstitute Phosphatase Inhibitor Cocktail III with 1mL of water.
Storage Upon receipt store at -20°C. It is stable for one year. Product is shipped on ice.
Equivalent Thermofisher (Product No. 78428, A32957)

Notes:

Type of Phosphatase Inhibitor CocktailSpecificityCatalog Number
Phosphatase Inhibitor Cocktail IITyrosine protein phosphatases, acid and alkaline phosphatasesAR1120
Phosphatase Inhibitor Cocktail IIISerine phosphatases, threonine phosphatases and protein tyrosine phosphatasesAR1121

BioChemicla Information

InhibitorInhibition Specificity
Sodium fluorideAcid phosphatase, Serine phosphatases, Threonine phosphatases
Sodium orthavanadiumAlkaline phosphatases, Tyrosine phosphatase
Sodium pyrophosphateSerine phosphatases, Threonine phosphatases
B-phosphoric acid glycerolSerine phosphatases, Threonine phosphatases

Assay Principle

Crude cell or tissue extracts contain a number of endogenous enzymes, such as proteases and phosphatases, which are capable of modifying the proteins present in the extracts. Inhibitors of these enzymes should be added to improve the yield of native proteins. Phosphatase inhibitor cocktail III protect phosphoproteins from serine phosphatases, threonine phosphatases and protein tyrosine phosphatases. The cocktail contains a mixture of four inhibitors, including sodium fluoride, sodium orthovanadate, beta-sodium glycerophosphate and sodium pyrophosphate. It is suitable for use with many kinds of cell and tissue extract, including primary cells, mammalian cells, animal tissue, plant tissue, yeast or bacterial cells.

Properties

Compatibility with reagents Fully compatible with cell lysis buffers and Broad Spectrum Protease Inhibitor Cocktail
Compatibility with assays Compatible with IEF/2D studies;
MS-compatible: not contain AEBSF;
Compatible with immobilized metal chelate affinity chromatography and 2D gel electrophoresis inhibitor components dialysis removal or desalting;
Reagent Type Western Blotting related reagent; Inhibitors
Usage To preserve phosphorylation state and protein functionality following cell lysis
Target Specificity Serine phosphatases, threonine phosphatases and protein tyrosine phosphatases
Target Sample Cell lysis extracts
Description Boster’s Phosphatase Inhibitor Cocktail III is a Western blot related that is to be added to cell lysis buffer to protect native phosphoproteins from dephosphorylation during proteins purification and sample preparation used in WB, Co-IP, and IP. 
Cite This Product Phosphatase Inhibitor Cocktail III (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR1121)
Application Animal tissue and cell extract with detergent
*Our Boster Guarantee covers the use of this product in the above tested applications.

Background

Phosphatase inhibitors are used when phosphorylation (activation) states of target proteins need to be studied and the phosphorylated residues of interest must remain intact. They are chemicals that aid in the extraction of intact proteins in their native modification state by inhibiting endogenous phosphatases that would otherwise dephosphorylate the proteins present in cell lysates and tissue extracts. Phosphatase Inhibitor Cocktail III contains individual components with specific inhibitory properties to provide an all- around protection of the protein phosphorylation state. 
Dynamic protein phosphorylation is a key cellular signaling mechanism for cell processes regulation. When tissues are lysed to make whole cell extracts, the loss of natural compartmentalization causes normal regulation of cellular signaling to get distorted, and resident cell phosphatases within the cell extract are free to disorderly dephosphorylate proteins. The usual consequence of this unregulated state is biologically meaningless representation of protein activities (i.e. phosphorylation status) and false negative staining in anti-phosphoprotein immunostaining analyses. The addition of phosphatase inhibitors to the cell lysis buffer aids in the preservation of phosphorylated residues at the time of cell disruption.

Features

• Dialyzable—inhibitors can be removed by dialysis, before performing assays known to be incompatible with phosphatase inhibitors.
• Supplied as a 100X concentrate—effective as 1X. For samples with high level of phosphatase may need 2-3X.
• Detergent compatible—can be used with lysis buffers and do not crossreact with detergents in the buffers.
• Protein Assay Compatible—works with BCA and commassie blue assays.

Usage and Handling

The product is supplied as white lyophilized powders. Since phosphatase levels may vary among cell and tissue types, it may be necessary to increase the concentration of inhibitors.

Protocol

1. Reconstitute Phosphatase Inhibitor Cocktail III in 1ml of double distilled water.
2. Add cocktail solution to extraction buffer in 1:100. (e.g., Add 10 μL of cocktail solution to 1mL extraction buffer.)
3. Proceed to extract protein fractions.
Notes:Aliquot and store reconstituted phosphatase inhibitor cocktail solution at -20˚C.

Phosphatase Inhibitor Cocktail III Images

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Phosphatase Inhibitor Cocktail III
Phosphatase Inhibitor Cocktail III
The degree of inhibition for protein, acid and alkaline phosphatase activity (% of activity) was measured in mouse brain extract following treatment with Boster Phosphatase Inhibitor Cocktail III.
Phosphatase Inhibitor Cocktail III
Isolate proteins in the absence (–) or presence (+) of Boster Phosphatase Inhibitor Cocktail III for PDGFR in mouse liver tissue extracts and ERK1/2 in NIH-3T3 cell extracts. Membrane and cytosolic proteins (20ug) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Incubate with primary antibodies. And Boster ECL substrate was used to generate the image.
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