BN-PAGE Protein Complex Isolation

H1299 cells were transfected using the Qiagen Effectene kit according to the manufacturer’s manual in a 10 cm dish, grown for about 24 h, harvested and collected in NP lysis buffer without detergent (20 mM Tris, 150 mM NaCl, 2 mM MgCl₂, 1 mM DTT, complete protease inhibitor cocktail (Roche), PhosSTOP (Roche) pH 7.4). Samples were lysed via three freeze and thaw cycles. After centrifugation, Benzonase (Merck Millipore) was added and incubated 1 h on ice. Samples were split and incubated with different urea concentration for 1 h on ice. An equal volume of NP lysis buffer was added containing the two-fold concentration of CHAPS (20 mM Tris, 150 mM NaCl, 2 mM MgCl₂, 40 mM CHAPS, 1 mM DTT, complete protease inhibitor cocktail (Roche), PhosSTOP (Roche) pH 7.4) and incubated for about 10 min. Afterwards, 3× NP sample buffer (60% Glycerol, 15 mM Coomassie G250) was added and the sample was loaded on the gel. BN-PAGE (3–12%, Thermo Fisher Scientific) was performed and blotted as previously described^{13 (link),35 (link)}.