4.1.1. tBid

Full-length murine Bid with a N-terminal His-tag and a cysteine to serine mutation at position 30 (leaving a single cysteine residue at position 126, and thus referred to as Bid 126C), was purified as previously described [10,28,43,76]. Briefly, a plasmid encoding the Bid 126C construct was transformed into *E. coli* BL21 arabinose inducible cells by electroporation. Single colonie large scale 5 L cultures in lysogeny broth (LB) were induced with 0.2% arabinose for 3 h at 37 °C. Cells were then spun down at 5000 g for 30 min at 4 °C and the pellet was resuspended in Bid lysis buffer: 10 mM HEPES pH 7, 100 mM NaCl, 10 mM imidazole, 1 mM PMSF, 10 μg mL−<sup>1</sup> DNase and 1× Halt Proteinase Inhibitor Cocktail (Thermo Fischer, Waltham, MA, USA). Cells were then vortexed, passed through a 16 gauge needle and lysed by at least 2 passes through a French Press. Cell debris were removed by centrifugation at 15,000 *g* for 30 min at 4 °C. The supernatant was incubated with 1.5 mL Ni-NTA agarose resin (Qiagen, Hilden, Germany) for 1.5 h at 4 °C with mixing. The slurry was added to a 20 mL column (Bio-Rad, Hercules, CA, USA) that retained the resin, after which the column was washed with 50 mL Bid wash buffer: 10 mM HEPES pH 7, 300 mM NaCl, 1% ( *w*/*v*) CHAPS (Bioshop Canada, Burlington, ON, Canada) and 10 mM imidazole. The protein was eluted in Bid elution buffer (10 mM HEPES pH 7, 100 mM NaCl, 0.1% ( *w*/*v*) CHAPS, 200 mM imidazole and 10% glycerol).

The purified Bid 126C was then fluorescently labelled with Alexa647 (Molecular Probes, now Thermo Fisher), which formed a covalent bond via maleimide chemistry to the cysteine residue at position 126 [43]. Briefly, proteins in Bid storage buffer (10 mM HEPES pH 7, 100 mM NaCl, 0.2 mM EDTA, 10% glycerol) supplemented with 0.5% *w*/*v* CHAPS were incubated with a 10-fold molar excess of dye for 2 h at room temperature and in the dark while under constant rotation. Unreacted free dye was then first quenched by addition of 1 mM DTT, then removed by gel filtration in a G-25 fine Sephadex column (GE Healthcare, Chicago, IL, USA). The labelling efficiency was determined from absorption measurements that gave both protein and dye concentrations, and found to be 70% in this case.

The fluorescently labelled Bid was then cleaved with Caspase-8 to obtain cleaved Bid (cBid), as described in Ref. [43]. The fluorescent label (at residue 126) is on the larger C-terminal fragment of the protein, referred to as truncated Bid (tBid). Since both the His-tag and the C30S mutation are on the small N-terminal fragment of Bid, the only difference between purified tBid-Alexa47 and wild type tBid is the presence of the fluorescent Alexa647 tag.
