Cell Signaling Technology

Product Pathways - Tyrosine Kinase / Adaptors

Src Antibody Sampler Kit #9935

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb #6943 40 µl W IP H M R 60 Rabbit IgG
Non-phospho-Src (Tyr527) Antibody #2107 40 µl W H M R (C) 60 Rabbit
Non-phospho-Src (Tyr416) (7G9) Mouse mAb #2102 40 µl W IP H M R (C) (X) 60 Mouse IgG2b
Phospho-Src (Tyr527) Antibody #2105 40 µl W IHC-P H M R (C) 60 Rabbit
Src (32G6) Rabbit mAb #2123 40 µl W IP H M R (C) 60 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
Anti-mouse IgG, HRP-linked Antibody #7076 100 µl Horse

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)
Reactivity Key:  H=Human  M=Mouse  R=Rat  C=Chicken  X=Xenopus
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each antibody in the kit detects endogenous levels of Src only when in the indicated phosphorylation state at the indicated residue. The antibodies may cross-react with other Src family members when in equivalent phosphorylation states.

Western Blotting

Western Blotting

Western blot analysis of extracts from pervanadate-treated (1 mM for 5 minutes) NIH/3T3 cells that have been stably transfected with a constitutively active form of Src (in which the regulatory tyrosine 527 residue has been mutated to phenylalanine), A431 cells and HepG2 cells, using Non-phospho-Src (Tyr416) (7G9 ) mAb #2102 (upper), v-Src antibody (middle) or Phospho-Src (Tyr416) Antibody #2101 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, using Phospho-Src (Tyr527) Antibody #2105 (A, B) or v-Src antibody (C, D). The phospho-specificity of the antibody was determined by treating the membrane with (B, D) or without (A, C) calf intestinal alkaline phosphatase (CIP) after Western transfer.

Western Blotting

Western Blotting

Western blot analysis of extracts from A431 cells, untreated or pervanidate-treated (1 mM, 15 minutes), using Non-phospho-Src (Tyr527) Antibody #2107 (upper) or v-Src antibody (lower).


Western Blotting

Western Blotting

Western blot analysis of cell extracts from various cell lines, using Src (32G6) Rabbit mAb #2123.

Western Blotting

Western Blotting

Western blot analysis of extracts NIH/3T3 cells, serum-starved or treated with human Platelet-Derived Growth Factor BB hPDGF-BB #8912 (100 ng/ml, 15 min), using Phospho-Src Family (Tyr416) (D49G8) Rabbit mAb #6943 (upper) or Src (36D10) Rabbit mAb #2109 (lower).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical staining of paraffin-embedded human breast carcinoma, showing membrane localization of phosphorylated Src, using Phospho-Src (Tyr527) Antibody #2105.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Src (36D10) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

IF-IC

IF-IC

Immunofluorescent staining of MCF-7 cells, using Src (36D10) Rabbit mAb #2109 alone (A), or the same antibody after a 1 hour preincubation with antigenic peptide (B).

Description

The Src Antibody Sampler kit provides an economical means of evaluating total Src protein levels and its phosphorylation status. The kit contains enough primary and secondary antibodies to perform four Western blot experiments per primary antibody.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic phospho- or non-phosphopeptides corresponding to residues surrounding Tyr527 of human Src protein. The mouse monoclonal antibody is produced by immunizing animals with a synthetic non-phosphopeptide corresponding to residues surrounding Tyr416 of human Src protein. The rabbit monoclonal antibody for P-Src Y416 is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr416 of human Src protein. The Src (32G6) rabbit monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino-terminus of human Src. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).

Lyn is a member of the Src family that is predominantly expressed in hematopoietic cells (3). Lyn participates in signaling from multiple cell surface receptors such as the B cell Ag receptor (BCR) and CD40 (4).Lck is essential for T-lymphocyte activation and differentiation (5,6). The activity of Lck is regulated by protein kinases and phosphatases. Phosphorylation of the C-terminal tyrosine 505 serves to downregulate Lck catalytic activity, while phosphorylation at tyrosine 394 leads to an increase in Lck activity (7).

  1. Thomas, S.M. and Brugge, J.S. (1997) Annu. Rev. Cell Dev. Biol. 13, 513-609.
  2. Hunter, T. (1987) Cell 49, 1-4.
  3. Yamanashi, Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6538-6542.
  4. Yamanashi, Y. et al. (1991) Science 251, 192-194.
  5. Molina, T. J. et al. (1992) Nature 357, 161-164.
  6. Straus, D. B. et al. (1992) Cell 70, 585-593.
  7. Chow, L. M. et al. (1993) Nature 365, 156-160.

Application References

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Protocols

Companion Products

Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.

For Research Use Only. Not For Use In Diagnostic Procedures.

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