RESEARCH USE ONLY

POLYCLONAL ANTIBODY Anti-Hck [pTyrpSer209/211] Phosphospecific Antibody, Unconjugated Code No. Isotype: Quantity: AT-7065 Rabbit IgG 100 µL (0.50 mg/mL)

BACKGROUND: Hck is a member of the Src family of non-receptor tyrosine kinases involved in the modulation of a wide variety of normal and pathogenic cellular processes including growth, development, differentiation, and carcinogenesis. These proteins contain a consensus structure comprised of one or more N-terminal SH2 and/or SH3 domains involved in protein-protein interactions, a central catalytic domain, and a short C-terminal domain. This C-terminal domain contains a tyrosine residue (tyrosine 522) that, when phosphorylated by Csk tyrosine kinase, functions as a negative regulatory site by providing a for the N-terminal SH2 domain. The association of these two domains blocks access to a critical tyrosine residue in the activation loop, the phosphorylation of which is required for activation. Release of this autoinhibition can be achieved either by dephosphorylation of tyrosine 522 or by phosphorylation of tyrosine 209 and/or serine 211 in the SH2 domain.

PRODUCT: Rabbit polyclonal immunoglobulin in Dulbecco’s phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), with 1.0 mg/mL BSA (IgG, protease free) as a carrier. 0.05% sodium azide.

IMMUNOGEN: The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of mouse Hck that contains tyrosine 207 and serine 209 (tyrosine 209 and serine 211 in human).

PURIFICATION: Purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated Hck protein. The final product is generated by affinity chromatography using a Hck-derived peptide that is phosphorylated at tyrosine 209 and serine 211.

SPECIFICITY: Rat Hck. Human (92% homologous) and mouse (100%) Hck have not been tested, but are expected to react. There is some cross-reactivity with Src family kinases and Lyn, but the antibody does not react with Src, Fyn, or Yes.

APPLICATIONS: The antibody has been used for Western blotting applications. For Western blotting applications, we recommend using the antibody at 0.35-1.0 µg/mL. At 0.50 µg/mL, the dilution provides 100 mL working solution, which at 10 mL/blot allows 10 blots to be performed. The optimal antibody concentration should be determined for each specific

15 B Constitution Way · Woburn, MA 01801 · Phone: 1.800.200.5459 · Fax: 781-939-6963 · www.mblintl.com RESEARCH USE ONLY

application. STORAGE: Store at –80ºC. Upon initial thawing, apportion into working aliquots and store at –80°C. Avoid repeated freeze-thaw cycles to prevent denaturing the antibody.

POSITIVE CONTROL: PC12 cells +/- sorbitol, Chick Embryo Fibroblast (CEF) cells expressing Src protein and plated on fibronectin, and recombinant activated Fyn, Lck and Lyn proteins.

REFERENCES: Young, M.A., et al. (2001) Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation. Cell 105(1): 115-126.

Hubbard, S.R. and J.H. Till (2000) Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 69: 373-398.

Thomas, S.M. and J.S. Brugge (1997) Cellular functions regulated by Src family kinases. Annu. Rev. Cell Devel. Biol. 13: 513-609.

Stover, D.R., et al. (1996) Modulation of the SH2 binding specificity and kinase activity of Src by tyrosine phosphorylation within its SH2 domain. J. Biol. Chem. 271(21): 12481-12487.

15 B Constitution Way · Woburn, MA 01801 · Phone: 1.800.200.5459 · Fax: 781-939-6963 · www.mblintl.com RESEARCH USE ONLY

Peptide Competition Extracts prepared from PC12 cells treated with 0.5 M sorbitol for 5 minutes were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4oC, then were incubated with 0.50 µg/mL Hck [pTyrpSer209/211] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1), the phosphopeptide immunogen (2), the non-phosphopeptide corresponding to the immunogen (3), a mix of generic phosphotyrosine and phosphoserine containing peptides (4), the phosphopeptide derived from the corresponding region of Yes (5), the phosphopeptide derived from the corresponding region of Fyn (6), the phosphopeptide derived from the corresponding region of Src (7), the phosphopeptide derived from the corresponding region of Lck (8) or, the phosphopeptide derived from the corresponding region of Lyn (9). After washing, membranes were

incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method.

The data show that the Hck [pTyrpSer209/211] antibody reacts only with phosphopeptides for the corresponding sequences of Hck, Lck, and Lyn, but not of Yes, Fyn, or Src, nor does it react with the corresponding Hck non-phosphopeptide nor with a mixture of generic pTyr and pSer phosphopeptides. These data demonstrate the selectivity of the antibody for Hck [pTyrpSer209/211] with some reactivity with the corresponding sequences of other members of the Hck/Lck/Lyn subfamily of Src kinases. Further experiments examining peptide concentration dependence demonstrate a preferential reaction with the Hck phosphopeptide over the corresponding Lck and Lyn phosphopeptides (not shown).

Multiple Extracts Control CEF cells (1), CEF cells expressing activated Src (2), spiked with recombinant activated Lck (3), Lyn (4), or Fyn (5), or, PC12 cells treated with 0.5 M sorbitol for 5 minutes (6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4oC, then were incubated with 0.50 µg/mL Hck [pTyrpSer209/211] antibody for two hours at room temperature in a 3% BSA-TBST buffer. After

washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method.

The data show that the Hck [pTyrpSer209/211] antibody reacts strongly with an endogenous protein in PC12 cells and more weakly with purified Lck and Lyn proteins, but does not recognize purified Src or Fyn proteins, demonstrating the specificity of the antibody.

15 B Constitution Way · Woburn, MA 01801 · Phone: 1.800.200.5459 · Fax: 781-939-6963 · www.mblintl.com