Product Pathways - Tyrosine Kinase/ Adaptors

Src Antibody Sampler Kit #9935

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Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken  Hm=Hamster  B=Bovine  X=Xenopus

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 blot analysis of extracts from COLO 201 cells with or without 24 hours starvation in serum-free medium (lanes 1 and 2), immunoprecipitates from COLO 201 cells using v-Src antibody treated with or without lambda phosphatase (lanes 3 and 4) and NIH/3T3 cells treated with PDGF (50 ng/ml) for 0, 2 and 5 minutes (lanes 5, 6 and 7, respectively), using Phospho-Src Family (Tyr416) Antibody #2101 (upper) and v-Src antibody (lower).

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 (top), v-Src antibody (middle) or Phospho-Src (Tyr416) Antibody #2101 (bottom).

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 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).

IHC-P (paraffin)

Immunohistochemical staining of paraffin-embedded human breast carcinoma, showing perinuclear localization of phosphorylated Src, using Phospho-Src Family (Tyr416) Antibody #2101.

IHC-P (paraffin)

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

IHC-FL (floating)

Immunohistochemical staining (confocal images) of rat brain hippocampus (dentate gyrus region), control and 4-hour reperfusion following 15 minute transient cerebral ischemia, using Phospho-Src Family (Tyr416) Antibody #2101. The arrows indicate activated microglia and arrowheads denote neurons. (Provided by Dr. Bingren Hu, University of Miami School of Medicine, Florida.)

IF-IC

Immunofluorescent analysis of NIH/3T3 cells, wildtype (A and C) or stably transfected with a constitutively active form of Src in which the regulatory Tyr527 residue has been mutated to phenylalanine (B and D), using Phospho-Src Family (Tyr416) Antibody #2101 (green), anti-tubulin antibody (red), and Hoechst stain (to visualize nuclei) (blue). Cells were serum-deprived for 4 hours prior to fixation.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic phospho- or non-phospho-peptides (KLH-coupled) corresponding to residues surrounding Tyr416 or Tyr527 of human Src protein. The mouse monoclonal antibody is produced by immunizing mice with a synthetic non-phospho-peptide (KLH-coupled) corresponding to residues surrounding Tyr416 of human Src protein. The rabbit monoclonal antibody is produced by immunizing rabbits with a synthetic peptide (KLH-coupled) corresponding to residues near the carboxy-terminus of human Src. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

The Src family of protein tyrosine kinases (including 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. Phosphorylation of Tyr416 in the activation loop of the kinase domain by Csk upregulates enzyme activity, whereas phosphorylation of Tyr527 in the carboxy-terminal tail 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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Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.