COL3A1 Antibody (B-10): sc-271249

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:100) -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 (1:100) -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

For Western blot analysis, vastus intermedius muscle was homogenized in RIPA buffer containing a protease inhibitor cocktail, PMSF, and sodium orthovanadate (Santa Cruz, SC‐24948). Homogenates were then centrifuged at 10,000 g for 10 min, and the supernatant was designated as Ext I. The pellets were resuspended in RIPA buffer containing 1% SDS and sonicated on ice, and the supernatant obtained by centrifugation was named Ext II (COL3A1 protein was detected in this fraction). The protein concentration of the supernatants was quantified with the Bio‐Rad Protein Assay Dye Reagent (Bradford reagent, #5000006). Protein aliquots (30 μg from each sample) were resolved on 1.0‐mm thick, 4%–12% gradient bis‐tris polyacrylamide gels (Invitrogen, NPO322PK2) and blotted onto nitrocellulose membranes (Bio‐Rad, #1704270). Membranes were blocked in 5% milk (Bio‐Rad, #1706404xtu) for 1 hr at room temperature, and all antibodies were diluted in this same solution. Membranes were incubated with primary antibodies recognizing COL3A1 (Santa Cruz, sc‐271249, 1:100) or β‐actin (Santa Cruz, sc‐47778, 1:2000) at 4°C for 16 hr. An HRP‐conjugated anti‐mouse secondary antibody (Kindle Biosciences, R1005, 1:3000‐20000) was incubated with the membrane for 45 min at 25°C. Positive bands were visualized using ECL reagent (Millipore, WBKLS0500).

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Manufacturer protocol

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