Pulverized muscle was added to ice-cold BN Lysis Buffer (ε-aminocaproic acid 500 mM, Imidazole 7.5 mM, EDTA 2 mM, NaCl 12 mM, glycerol 10%, DDM 1%, PMSF 1 mM) and protease inhibitor cocktail (Roche).
Pellets were resuspended in the same buffer containing 20 mM DDM (1% (w/v)), 6 mM dodecyl octaethylene glycol monoether (C12E8, Sigma) (0.32% (w/v)) and 100 µM Tris 2-carboxyethyl phosphine (Tris 2-carboxyethyl phosphine
The spectra of absorbance and fluorescence of AF488-MscL in PBS + DDM (1 mM DDM) were measured using Agilent 8453 UV-Vis absorbance spectrophotometer (Agilent Technologies, Santa Clara, CA) and PC1 spectrofluorimeter (ISS, Inc., Champaign, IL), respectively.
Three detergents [1% DDM, 1% C12E8 (octaethylene glycol dodecyl ether) and 1% Triton X-100] were deemed to be quite effective, at least for bait extraction, as determined by Western blotting using an anti-FLAG antibody that detects the SPA tag (Fig. 6a).
The assembled Bam(AB)(CDE) complex was eluted with 3.5 ml buffer A containing 500 mM Imidazole and injected into an S-200 column equilibrated with TBS pH 8, 0.03% n-dodecyl-β-D-maltoside (DDM), 1 mM tris 2-carboxyethyl phosphine (tris 2-carboxyethyl phosphine
Membranes were thawed on ice, resuspended to a total protein concentration of 10 mg/mL in buffer A, solubilized via addition of 1% DDM, 1 mM PMSF, and Roche protease inhibitor cocktail (no EDTA), and stirred at 4 °C for 1 h.
