Immunoprecipitation of extracts from NIH/3T3 cells, untreated (-) or sodium vanadate-treated (1 mM, 0.5 hr; +), using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Sepharose® Bead Conjugate), followed by western blot analysis using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411.
Do not use for IAP in the PhosphoScan® method. IAP immobilized antibody is specific to the PhosphoScan® Kit.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Sepharose® Bead Conjugate) is useful for immunoprecipitation of tyrosine-phosphorylated proteins. P-Tyr-100 is immobilized via the conjugation of carbohydrates to cross-linked agarose hydrazide beads. The antibody does not cross-react with proteins phosphorylated on serine or threonine. (U.S. Patent No's.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)
All Species Expected
Monoclonal antibody is produced by immunizing animals with phospho-Tyr-containing peptides.
Tyrosine phosphorylation plays a key role in cellular signaling (1). Research studies have shown that in cancer, unregulated tyrosine kinase activity can drive malignancy and tumor formation by generating inappropriate proliferation and survival signals (2). Antibodies specific for phospho-tyrosine (3,4) have been invaluable reagents in these studies. The phospho-tyrosine monoclonal antibodies developed by Cell Signaling Technology are exceptionally sensitive tools for studying tyrosine phosphorylation and monitoring tyrosine kinase activity in high throughput drug discovery.