paxillin Antibody (177): sc-136297

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:200) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:200) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

Proteins were extracted using a Triton based buffer (0.5 % Triton, 300 mM Sucrose, 100 mM NaCl, 10 mM PIPES, 3 mM MgCl2*6H2O, 5 mM EDTA) supplemented with 1 μM DTT, 1 μM PMSF, 1 μM Pepstatin, 1 μg/ml Aprotenin, 0.5 μg/ml Leupeptin and phosphatase inhibitor cocktail 2 (Sigma, Cat. No. P5726). Following 10 min on ice the lysate was centrifuged at 14,000 rpm and the pellet discarded. Antibodies used for detection in western blot are as follows: Anti-phospho EGFR Y1092 (Abcam, 1:1000 Cat. No. ab40815), Anti-EGFR (Abcam, 1:1000 Cat. No. ab2430), anti-phospho AKT S473 (Cell signaling, 1:1000, Cat. No. 4058), Anti-AKT (Cell signaling, 1:1000, Cat. No. 11E7), anti-paxillin (Santa Cruz, 1:200, Cat. No. sc-136297), anti-beta-Actin (Abcam, 1:1000,Cat. No. ab6276), Strepavidin coupled HRP (Jackson Immunoresearch Laboratories, 1:1000). Secondary antibodies coupled to horseradish peroxidase (Jackson Immunoresearch Laboratories).

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