ATP5A Antibody (51): sc-136178

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:2000) -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 (1:2000) -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

HCECs, MCECs, and isolated mitochondria from HCECs were lysed in lysate buffer (in mM: 25 HEPES, 150 NaCl, 10 MgCl2, and 1 EDTA, with 1% IGEPAL, 1% protease inhibitor, and 1% phosphate inhibitor, pH 7.5). Proteins were separated with a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Blots were incubated with the following primary antibodies: anti-Cx40 (Santa Cruz, sc-20466; 1:2,000), anti-ATP5A (Santa Cruz, sc-136178; 1:2,000), anti-Actin (Santa Cruz, sc-1616; 1:4,000), anti-Cx37 (Thermo Fisher Scientific, 42-4400; 1:1,000), anti-Cx43 (Thermo Fisher Scientific, 71-0700; 1:2,000), anti-Cx45 (Thermo Fisher Scientific, 41-5800; 1:2,000), or anti-GAPDH (Thermo Fisher Scientific, MA5-15738; 1:4,000), followed by horseradish peroxidase-linked secondary antibody incubation. The immunoblots were identified with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34087). Band intensity was calculated with ImageJ 1.48v (National Institutes of Health), and the intensity data from focus proteins were normalized to GAPDH or Actin (for whole cell and cytosolic samples) or ATP5A (for mitochondrial samples) and are expressed in arbitrary units.

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