Anti-LC3 (Rat) pAb

Protocol tips

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This antibody reacts with LC3-I (18kDa) and LC3-II (16 kDa) on Western blotting	1:500 dilution of primary antibody

Upstream tips
This antibody reacts with LC3-I (18kDa) and LC3-II (16 kDa) on Western blotting
Protocol tips
1:500 dilution of primary antibody

Publication protocol

Total cellular protein extracts were prepared by scraping the cells into modified RIPA buffer (50 mM Tris HCl (pH 7.4), 150 mM NaCl, 0.25% sodium deoxycholate, 1% Igepal, 1 mM EDTA, 1x Pefabloc, 1x protease inhibitor cocktail, 1 mM Na3VO4, 1 mM NaF). All protein samples were separated on NuPAGE 4–12% Bis-Tris gels and electrophoretically transferred onto PVDF membrane (Invitrogen Life Technologies NP0322 and IB401001). Membranes were incubated with anti-LC3 (polyclonal rabbit antibody–MBL PD014, 1:500 dilution), anti-LAMP 1 (monoclonal mouse antibody–Abcam ab25245, 1:5000 dilution), anti-LAMP 2 (polyclonal rabbit antibody—Abcam ab101325, 1:500 dilution) or anti-cathepsin B (mouse monoclonal antibody–Abcam ab58802, 1:500 dilution) antibodies overnight at 4°C and with anti-β-actin (loading control) (Sigma A5441) for one hour at room temperature. Proteins were visualized using relevant IR-Dye conjugated secondary antibodies (Rockland) on the Odyssey IR imaging system (Li-Cor, Cambridge, United Kingdom).

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