Immunoprecipitation of phospho-tyrosine proteins from A-431 cell extracts, untreated (-) or treated with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml, 5 min; +), using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423 (lanes 3 and 4), or Phospho-Tyrosine (P-Tyr-1000) MultiMab™ Rabbit mAb mix (Sepharose® Bead Conjugate) (Lanes 5 and 6). Western blot analysis was performed using Phospho-Tyrosine (P-Tyr-1000) MultiMab™ Rabbit mAb mix (HRP Conjugate) #12695. Lanes 1 and 2 are 10% input.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads.
Phospho-Tyrosine (P-Tyr-1000) Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of phospho-tyrosine containing proteins and peptides. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Tyrosine (P-Tyr-1000) MultiMab™ Rabbit mAb mix #8954.
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 (P-Tyr-1000) MultiMab™ Rabbit mAb mix (Sepharose® Bead Conjugate) recognizes a broad range of tyrosine-phosphorylated proteins and peptides. This antibody does not cross-react with proteins or peptides containing phospho-Ser or phospho-Thr residues.
Human, All Species Expected
MultiMab™ rabbit monoclonal mix antibodies are prepared by combining individual rabbit monoclonal clones in optimized ratios for the approved applications. Each antibody in the mix is carefully selected based on motif recognition and performance in multiple assays. Each mix is engineered to yield the broadest possible coverage of the modification being studied while ensuring a high degree of specificity for the modification or motif.
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.