Dithiothreitol (dtt)

The plasmid carrying synthetic genes with Escehrichia coli (E. coli) optimized codons for A $\beta$ 42 wild-type (PetSac, cloned by us [22 (link)]) as well as S8C, Y10C, S26C, V40C, and A42C (Pet3a, purchased from Genscript) were transformed into Ca ${}^{2+}$ competent cells of E. coli strain BL21 DE3 pLysS star and the protein was expressed in auto-induction medium [23 (link)]. The peptides were purified using ion exchange chromatography (IEX) as described with the minor change that lower salt concentration (50 mM NaCl, (Duchefa Biochemie, CAS no. 7647-14-5)) was used to elute the peptides, and size exclusion chromatography (SEC) on a 26 × 600 mm Superdex 75 column was used instead of spin filters for base on molecular size. The ion exchange and SEC buffers contained 1 mM dithiothreitol (DTT, PanReac Applichem, CAS no. 3483-12-3) to avoid dimerization of cysteine mutants. The final SEC was performed in buffer without DTT in order to isolate monomer and remove DTT from the sample prior to adding the label. The purified monomeric peptides were lyophilized as aliquots until further use.

Formation of Bax-dimers or Bak high-molecular-weight complexes was analyzed using bismaleimidohexane (BMH) (Thermo Fisher Scientific, #22330) as described [60 (link)]. Briefly, mitochondrial pellets either isolated from staurosporine-treated Bak KO HeLa cells, ABT-737 + s63845-treated Bax KO HeLa cells or upon tBid-treatment were resuspended in crosslinking buffer (20 mM HEPES [pH 7.5], 100 mM sucrose, 2.5 mM MgCl2, and 50 mM KCl) and were then treated with 0.5 mM BMH for 30 min at room temperature in the dark. Crosslinking was stopped by adding 0.5 mM dithiothreitol (DTT) (Panreac AppliChem, #A2948.0025), and samples were heated at 95 °C for 5 min and analyzed by Western blotting.