Recombinant Anti-VDAC1 / Porin antibody [EPR10852(B)] - Mitochondrial Loading Control (ab154856)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:2000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:2000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Hippocampal mitochondria from copper-treated and untreated mice were extracted with lyse mitochondrial buffer with 2% CHAPS in Tris buffered saline (TBS; eg, 25 mM Tris, 0.15 M NaCl; pH 7.2, Product No. 28379, Thermo Fisher Scientific, USA). Hippocampal nuclear extract from each group was extracted with lysis buffer (Beyotime, Haimen, China) with phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA). The concentration of total proteins was measured with the BCA protein assay kit (Thermo Fisher Scientific, USA). The protein samples were mixed with loading buffer and heated for 8 min at 100°C, then separated on 12% SDS-PAGE and transferred to PVDF membranes. Membranes containing the transferred proteins were blocked with 5% skim milk in TBST (150 mM NaCl, 10 mM Tris, 0.1% Tween-20, pH 8.0). The blocked membranes were incubated with anti-β-actin (1:1000, Santa Cruz, CA, sc-47778), anti-VDAC1 (1: 2000, Abcam, ab154856), anti-synapsin II (1: 5000, Abcam, ab76494), anti-COX5B (1:1000, Abcam, ab180136), anti-QCRI (1: 1000, Abcam, ab131152), anti-IDH3A (1: 1000, Abcam, ab58641), anti-PGAM1 (1: 1000, Abcam, ab184232), anti-PDIA3 (1: 2000, Abcam, ab10287), anti-GRP75 (1: 500, Santa Cruz, sc-133137), and anti-SODC (1: 5000, Abcam, ab13498) in TBST buffer overnight at 4°C. After washing in TBST, the membranes were incubated with a 1: 3000 dilution of anti-rabbit or anti-mouse IgG HRP secondary antibody diluted in TBST for 1 h. Subsequently, the membranes were washed in TBST (4 × 10 min) and developed using chemiluminiscence reagents from an ECL kit (Thermo Scientific Pierce ECL, USA). The blots were detected on a phosphorimager and analyzed using ImageQuant 1D software

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-VDAC1 / Porin antibody [EPR10852(B)] - Mitochondrial Loading Control (ab154856) below.

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