LC3A/B (D3U4C) XP® Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in RIPA buffer containing 1 mM PMSF	Dilute primary antibody at 1:400, 4 °C overnight.

Upstream tips
Lyse cells in RIPA buffer containing 1 mM PMSF
Protocol tips
Dilute primary antibody at 1:400, 4 °C overnight.

Publication protocol

Western blot was used to detect apoptosis and autophagy related proteins. After treatment, the cells were collected and washed with cold PBS, lysed in RIPA buffer containing 1 mM PMSF, the lysates were then centrifuged at 12,000× g for 15 min at 4 °C. The quantification of total protein was made by using a BCA Protein Assay Kit (BestBio, Shanghai, China) and was separated by 10–15% SDS-PAGE, which was then transferred to polyvinylidene difluoride membranes (Millipore, Temecula, CA, USA). The membranes were blocked with 5% non-fat dry milk in PBS-Tween 20 for 2 h, and were incubated with a polyclonal rabbit anti-human cleaved-caspase3, PARP, p62, LC3B antibody (1:400) at 4 °C overnight. The membranes were incubated with a secondary HRP-conjugated anti-rabbit antibody (1:1000) for 2 h. The immunoreactivity bands were visualized by chemiluminescence.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A/B (D3U4C) XP® Rabbit mAb below.

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