Anti-COX IV antibody - Mitochondrial Loading Control (ab16056)

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 (1:1000) -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 (1:1000) -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

Mouse liver and HepG2 cells were lysed in radioimmunoprecipitation buffer (Sigma, UK) with a protease inhibitor cocktail (Roche, UK). Mouse liver extracts were taken to a final concentration of 10 mM MgCl2, 10 mM CaCl2 and DNase treated with 50 units of DNase-I (Promega, UK) at 37 °C for 2 h. Subsequent heat inactivation was performed at 65 °C for 30 min. Protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce, UK). Protein lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, UK). Membranes were blocked in 5% (w/v) milk/TBST for 1 h prior to incubation with primary antibody in TBS-Tween20 (T) for 12–16 h at 4 °C. The following antibodies were used: anti-L-FABP (1:4000; ab7847; Abcam, UK); anti-COXIV (1:1000; ab16056; Abcam, UK) and anti-TUBA1A (1:10,000; ab7291; Abcam, UK). Anti-GLO1 (1:1000; ab96032; Abcam, UK) was used for mouse liver samples and anti-GLO1 (1:4000; SAB4200193; Sigma UK) was used for HepG2 extracts. The membranes were washed in TBST and incubated with HRP-conjugated secondary antibodies (Sigma, UK) for 2 h or fluorescently-tagged antibodies (LICOR Biosciences, UK) for 1 h. Proteins were either visualized by enhanced chemiluminescence (Pierce, UK) and exposed to Hyperfilm™ (GE Healthcare, UK), or by infrared fluorescence using a LI-COR Odyssey imaging system (LI-COR Biosciences, UK). Densitometry was performed using ImageJ [60] or Image Studio (LI-COR Biosciences, UK) software.

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Manufacturer protocol

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