5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB)-mediated chemical labelling of 3M3SH

To quantify free 3M3SH, a 75 µl volume from each reaction (isolated as described above) was taken at the appropriate time point and centrifuged at 13, 000 rpm for 2 min. 950 µl labelling solution, consisting of: 800 µl dH₂O, 100 µl UltraPure Tris (pH 8.0) and 50 µl DTNB (Ellman, 1958) stock solution (50 mM sodium acetate, 2 mM DTNB, dissolved in dH₂O) was added to a 1.5 ml disposable cuvette. 50 µl reaction supernatant was added to the cuvette, mixed by pipetting and incubated at room temperature for 5 min. The A_412nm was recorded on a Jenway 6305 spectrophotometer. (Van Horn, 2003). Where appropriate, E. coli strains harbouring inducible genes on the pBADcLIC2005 plasmid were pre-induced during overnight growth with 0.0001% L-arabinose. In all experiments cells only (no added substrate) and substrate only (no added cells) were also included.