Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748)

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.	-Mouse (1:5000) -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
-Mouse (1:5000) -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

Cells were homogenized by sonication in 2x Laemmli buffer containing urea (Sigma) and DTT (Sigma) to a final concentration of 0.8M and 50 mM respectively. Afterwards the homogenates were boiled at 99°C for 5 min. The indicated proteins were separated with 8% polyacrylamide gels and overnight wet transfer, or on 10% or 12% mini protean TGX stain-free gels (Bio-Rad). Stain-free gels were activated and imaged with the ChemiDoc imager (Bio-Rad) before transfer to PVDF membranes using the Trans Blot Turbo System (Bio-Rad). The membranes were blocked in 5% fat free milk for 1 hr at room temperature and rinsed in PBS-Tween 20. Incubations with primary antibodies were done overnight at 4°C followed by incubations with secondary antibodies for 1.5 hr at room temperature. The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 (Vonk et al., 2017), VPS13C (Sigma). Membranes were developed using ECL reagent (Thermo Fisher Scientific) and the signal was visualized using the ChemiDoc imager (Biorad), images exported as. tiff files and densitometric analysis of band intensities was performed using ImageJ software.

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Manufacturer protocol

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