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Synthetic phosphopeptide derived from the region of human c-Met that contains tyrosines 1230, 1234 and 1235. (Peptide available as ab41697.)
Binding of scatter factor (SF)/hepatocyte growth factor (HGF) to the c Met receptor tyrosine kinase (RTK) triggers receptor dimerization and phosphorylation on multiple residues within the juxtamembrane, catalytic core and cytoplasmic tail domains, thereby regulating receptor internalization, catalytic activity and multisubstrate docking. c Met contains three tyrosines (Tyr-xx- x-Tyr-Tyr motif) within the activation loop of the catalytic domain. This is also seen with the insulin receptor, insulin-like growth factor receptor (IGF1) receptor and nerve growth factor (NGF) receptors/Trks, for which phosphorylation of all three tyrosines is required for full activation. With c Met (and the related family member, RON) phosphorylation of tyrosines 1234 and 1235 has been shown to be important in receptor activation. Activation of the c Met receptor results in binding and/or phosphorylation of many intracellular signaling proteins including multiple adaptor proteins (e.g., Grb2, Shc, Cbl, Crk, cortactin, paxillin, and GAB1), and a variety of other signal transducers (e.g., PI 3-kinase, FAK, Src, Erk1&2, JNK, PLC-ã, and STAT3).
Our Abpromise guarantee covers the use of ab5662 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration.|
|ICC/IF||Use at an assay dependent concentration.|
|WB||1/1000. Detects a band of approximately 169 kDa.|
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse colon tissue sections labeling Met (c-Met) (phospho Y1230 + Y1234 + Y1235) with ab5662 at 1/100 dilution. the tissue was fixed with paraformaldehyde and heat-mediated antigen retrieval was performed using a citrate buffer. The sample was blocked with 3% BSA for 30 hours, followed by staining with ab5662 at 1/100 in 3% BSA/PBS for 12 hours at 4°C. An Alexa Fluor® 488 conjugated donkey anti-rabbit IgG secondary antibody was used at 1/1000 dilution.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse small intestine tissue sections labeling Met (c-Met) (phospho Y1230 + Y1234 + Y1235) with ab5662 at 1/200 dilution. The tissue was fixed in 10% formalin and heat-mediated antigen retrieval was performed using 10 mM citrate buffer for 5 minutes in a pressure cooker. A biotinylated goat anti-rabbit secondary antibody was used at 1/250 dilution.
Peptide Competition: Extracts prepared from 293T cells left unstimulated (1) or stimulated with HGF (2-5) 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 4°C, then were incubated with the ab5662 antibody, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 antirabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that only the phosphopeptide corresponding to c Met [pYpYpY1230/1234/1235] block the antibody signal, demonstrating the specificity of the antibody. Note: There are three isoforms of c Met, two of which are recognized by this antibody.
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