p-NOS3 Antibody (pT495.33): sc-136519

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1000) -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 (1:1000) -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

NOX associated key proteins (p67Phox, p47Phox, and p-p40Phox) and p-eNOS was analyzed as previously described using the Western-blot method [50,56]. In the present study, β-actin, GAPDH, and Na+/K+-ATPase were used as a corresponding standard protein markers of cytoskeleton, cytosol, and cell membrane. Each P2 fraction (mentioned above) was mixed with corresponding PMS and diluted 1:1 (v/v) in lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail). All the samples were centrifuged at 13,000g for 10 min at 4°C and then collected supernatants further centrifuged for 1h at 100,000g at 4°C. The obtained pellets and supernatants were designated as the membrane and cytosolic fraction [48]. Equal amounts of protein (50μg) were loaded with the appropriate marker (Bio-Rad) on a gradient gel (4-20% Tris-HCl), followed by transfer to a nitrocellulose membrane using a semi-dry transfer system (Bio-Rad) in transfer buffer (25 mM Tris, 150 mM glycine, 20% MeOH) for 2 h at 15 volt. NOX protein analyzing membranes were blocked with 5% milk, and p-eNOS assessing blots were blocked with 3% bovine serum albumin (BSA), in Tris/saline buffer-Tween (TBST). Primary antibodies of p-67Phox, p-47Phox, p-40Phox, p-eNOS β-actin, GAPDH, and Na+/K+-ATPase were added at a concentration of 1:1000 and incubated overnight at 4°C. The blots were washed three times in TBST and incubated for 1 h with alkaline phosphatase conjugated secondary antibodies in a 1:8000 dilution. All the membranes were washed three times in TBST for 10 min and developed in Sigma Fast tablets (BCIP/NBT substrate). Blots were then dried, scanned with Adobe Photoshop, and quantified with Scion Image (PC version of Macintosh-compatible NIH Image).

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