Antibody Customer Review for Phospho-RAF-1 (S301) Polyclonal Antibody (STJ90595)

Target information

Raf-1 provides the regulatory link between the membrane-associated Ras GTPases and the Mitogen-Activated Protein Kinase (MAPK) cascade pathway for cell proliferation, differentiation and survival. This switch is key for oncogenic transformation. Once phosphorylated this serine/threonine kinase phosphorylates MEK (a MAPK) which cascades the signal onwards.

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1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: Phospho-Raf-1 (S301) Polyclonal Antibody (STJ90595)
2. WHAT WAS USED? Primary Antibody: Phospho-Raf-1 (S301) Polyclonal Antibody (STJ90595) Provider: St John’s Laboratory Dilution ratio: 1:500 Application: Western Blot Materials for validation: A549 & NIH3T3 cells
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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