Atg12 (D88H11) Rabbit mAb

Protocol tips

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	For Western Blotting dilute at 1:1000 and for Immunoprecipitation at 1:100. Incubate overnight at 4°C

Protocol tips
For Western Blotting dilute at 1:1000 and for Immunoprecipitation at 1:100. Incubate overnight at 4°C

Publication protocol

Cells were lysed in RIPA (1% Triton X-100 (Sigma, T8787), 1% sodium deoxycholate (Sigma D6750), 0.1% SDS (Sigma, L6026), 25 mM Tris, pH 7.6, 150 mM NaCl, 10 mM NaF (Sigma, RPS6776), 10 mM β-glycerophosphate (Sigma, G9422), 1 mM Na3VO3 (Sigma, G9422), 10 nM calyculin A (Sigma, C5552), and protease inhibitors cocktail (Sigma, P8340)). Lysates were cleared by centrifugation for 15 min at 4°C, boiled in sample buffer, resolved using SDS-PAGE, and transferred to PVDF membrane (Bio-Rad, 162–0177). The membranes were blocked, incubated with the primary antibodies indicated overnight at 4°C, washed, incubated with HRP-conjugated secondary antibodies (Fisher Scientific, NC9832458; Fisher Scientific, NC9889490; Jackson ImmunoResearch Laboratories, 711–035–152), and analyzed by enhanced chemiluminescence (ECL, Pierce PI-32109). The anti-LC3 antibody generated in our laboratory has been described previously and is now commercially available (EMD Millipore, ABC232).35 Other antibodies used for immunoblotting include: SQSTM1 (Progen, GP62-C), NBR1 (Novus, NBP1–71703), ATG12 (Cell Signaling Technology, 4180), ATG5 (Novus, NB110–53818), TUBA (Sigma, T6199) and GAPDH (Santa Cruz Biotechnology, sc-137179).

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Atg12 (D88H11) Rabbit mAb below.

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