Anti bim

Interactions between Mcl-1 and Bim or Bak were evaluated by co-IP analysis. CHAPS buffer (150 mmol/L NaCl, 10 mmol/L HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid] pH 7.4, protease inhibitors, and 1% CHAPS) was employed to avoid artifactual associations [43] (link). Cells were lysed in CHAPS buffer and 200 µg of protein per condition were immunoprecipitated with 1 µg anti-Mcl-1 (Santa Cruz Biotechnology or BD Biosciences), anti-Bak, or anti-Bim (Santa Cruz Biotechnology), followed by Dynabeads (Dynal, Oslo, Norway). IP samples were then subjected to Western blot analysis using anti-Bim (Millipore), anti-Mcl-1, or anti-Bak (Santa Cruz Biotechnology) as primary antibodies, respectively.
To monitor Bak and Bax conformational change, anti-Bax (6A7, Sigma) or anti-Bak (Ab-1, Millipore) antibodies, which only recognize Bax or Bak that have undergone conformational change, were used for IP, followed by Western blot analysis using anti-Bax and anti-Bak as primary antibodies.

Cells were lysed either in 8 M Urea or in RIPA buffer (Thermo Fisher, #89900) and boiled in Laemmli-buffer containing DTT. Samples were heated for 5 min. Antibodies used were: Bak(NT), Bak(aa23-38), Bak(Ab-1), Bak(G23) as above. The following antibodies were purchased from Cell Signaling unless indicated otherwise: anti-Bim (# C34C5), anti-GAPDH (Millipore, #MAB374), anti-Bcl-X_L (#54H6), anti-Bcl-2 (#2870), anti-Bax (#2772), anti-VDAC (#4661), anti-Hsp60 (#4870), anti-Mcl-1 (BD, #559027), anti-Hsp60(Ctr) (Enzo Life Sciences, #ALX-804-072), anti-Tom22 (Santa Ccruz, sc-58308), anti-Bcl-w (#2724 S), anti-GFP (Roche, #11814460001), anti-cytochrome c (#11940) and anti-tBid [64 (link)], anti-BiP (Cell signalling, #3177), anti-VDAC2/3 (Thermo Fisher, #PA141205). Peroxidase-conjugated secondary antibodies were goat anti-rabbit IgG (Sigma, #A6667), goat anti-rabbit Fc (Sigma, #AP156P), goat anti-mouse IgG (Dianova, #115035166), goat anti-mouse Fc (Sigma, #AP127P) and goat anti-rat IgG (Dianova, #112035062).