Atg7 (D12B11) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’	Primary antibody is together treated with protein A/G Plus-agarose immunoprecipitation reagent at 4 °C for 3 h or overnight.

Upstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’
Protocol tips
Primary antibody is together treated with protein A/G Plus-agarose immunoprecipitation reagent at 4 °C for 3 h or overnight.

Publication protocol

Immunoprecipitation and immunoblot analysis were performed as described previously41. In short, HEK293T, Hela, THP1 or MT2 cells were transfected with various combinations of plasmids or siRNA. At 24 h after the transfection, the cell lysates were prepared in lysis buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’ (Roche). After centrifugation for 20 min at 14,000 g, supernatants were collected and incubated with the indicated antibody together with protein A/G Plus-agarose immunoprecipitation reagent (sc-2003, Santa Cruz Biotechnology) at 4 °C for 3 h or overnight. After three washes, the immunoprecipitants were boiled in SDS sample buffer for 10 min and analyzed by immunoblot. For endogenous coimmunoprecipitation experiments, the cell lysates of MT2 cells or THP1 cells were incubated with indicated antibodies and analyzed by immunoblot.

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Manufacturer protocol

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