Immunoprecipitation of Jurkat cell extracts, untreated or pervanadate-treated, using Mouse IgG (Magnetic Bead Conjugate) #5873 (lanes 1 and 2) or Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Magnetic Bead Conjugate) (lanes 3 and 4). The western blot was probed using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate) #5465.
This Cell Signaling Technology (CST) antibody is immobilized by the covalent reaction of hydrazinonicotinamide-modifed antibody with formylbenzamide-modified magnetic bead. Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Magnetic Bead Conjugate) is useful for immunoprecipitation assays. The unconjugated Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 reacts with all species of phospho-tyrosine protein. CST expects that Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Magnetic Bead Conjugate) will also recognize phospho proteins in all species.
Supplied in PBS Buffer (pH 7.2), 0.1% Tween® 20. Store at 4°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: 408
Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Magnetic Bead Conjugate) contains high affinity beads. ELISAs against a wide variety of phosphopeptides indicate that P-Tyr-100 binds phospho-Tyr in a manner largely independent of the surrounding amino acid sequence. 2D gel western blot analysis of pervanadate-treated cell extracts shows that P-Tyr-100 interacts with a broad range of tyrosine-phosphorylated proteins. P-Tyr-100 does not cross-react with peptides containing phospho-Ser or phospho-Thr. (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-tyrosine 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.
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