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c-SRC is a non-receptor tyrosine kinase, which is activated following stimulation of various cell receptors (E.g. GPCR¡¯s, integrins and receptor tyrosine kinases). This molecule has diverse range of interactions which form a network of channels for intracellular communication, which makes its role difficult to specify. Although, SRC appears to take on a primary role in activating other protein tyrosine kinases. Where receptors dimerise or cluster SRC is recruited, and phosphorylates tyrosine residues within the cytoplasmic domains. As such this gives c-SRC its proto-oncogene status as its elevated expression links with cancer expression as other signals are promoted.
1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: Phospho-c-Src (S75) Polyclonal Antibody (STJ90958)
2. WHAT WAS USED? Primary antibody: Phospho-c-Src (S75) Polyclonal Antibody (STJ90958) Provider: St John’s Laboratory Dilution ratio: 1:500 Application: Western Blot Materials for validation: LOVO cell line
3. WHAT WAS THE PROTOCOL? Treatment of materials: Cells were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 1% SDS, supplemented with protease inhibitors (PMSF 1mM, Leupeptin 1ug/ml, Aprotinin 1ug/ml, Pepstatin 1ug/ml). Proteins were reduced and denatured by heating at 100¡æ for 5 min with subsequent cooling before use. Gel electrophoresis: 60ug/lane Transfer information: Nitocellulose membrane. Blocking: Blocked with 5% BSA for 0.5-1hr Primary Ab Application: Overnight at 4oC, 5% BSA in TBST. Secondary Ab Application: After washing with TBST 3 times membrane incubated secondary antibody for 1h at room temperature. Visualization: ECL kit
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