COX4 Antibody (F-8): sc-376731

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:5000) -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 (1:5000) -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 from cultured cells using cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich). The soluble protein solutions were mixed with 4X sample buffer (0.25 M Tris-HCl, 20% mercaptoethanol, 8% SDS, 20% sucrose, 0.008% bromophenol blue; pH 6.8) and boiled for 5 min. Equal quantities of protein (20 µg) were loaded onto SDS-PAGE (10% separation gel and 5% spacer gel). The separation gel was then run at 80 V for 15 min and the spacer gel was run at 120 V for 1 h with 1X running buffer (25 mM Tris-HCl, 200 mM glycine, 0.1% (w/v) SDS; Bio-Rad, Hercules, CA, USA). The gel was then transferred onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) at 80 V for 3 h in an ice-cold environment. Following blocking in 5% skimmed milk in Tris-buffered saline with Tween-20 (TBST; 20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween-20), the membranes were incubated with the following primary antibodies at 4°C overnight: Mouse monoclonal anti-human B cell lymphoma 2 (Bcl-2; 1:2,000; cat. no. sc-7382; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); mouse monoclonal anti-human Bcl-2-associated X protein (Bax; 1:3,000; cat. no. sc-20067; Santa Cruz Biotechnology, Inc.); mouse monoclonal anti-human cytochrome c (1:2,000; cat. no. sc-514435; Santa Cruz Biotechnology, Inc.); mouse monoclonal anti-human cytochrome c oxidase subunit IV isoform 1 (COX4; 1:5,000; cat. no. sc-376731; Santa Cruz Biotechnology, Inc.); rabbit polyclonal anti-human cleaved caspase-3 (1:1,000; cat. no. sc-22171-R; Santa Cruz Biotechnology, Inc.); mouse monoclonal anti-human β-actin (1:10,000; cat. no. sc-130065; Santa Cruz Biotechnology, Inc.). The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) or HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) secondary antibodies for 1 h at room temperature. The membranes were washed three times with TBST and the proteins were detected with the enhanced chemiluminescence system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The relative densities of protein bands were analyzed using Image J 2.1 software (National Institutes of Health, Bethesda, MA, USA).

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Manufacturer protocol

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