Prestained page ruler

Extracellular proteins were obtained from bacterial supernatants following the protocol described by Sánchez et al. [17 (link)]. Briefly, trichloroacetic acid (TCA) was added to the spent supernatants at a final concentration of 6% (w/v) and incubated ON at 4°C. Samples were then centrifuged and washed twice with ice-cold acetone (10,000 ×g, 4°C, 10 min). Pellets were dried for one hour at 37°C in a heat block, and the precipitate corresponding to 50 mL of culture was resuspended in 200 μL of Laemmli buffer 5X [18 (link)] with the help of an ultrasound bath (15 min) (Ultrasons-H, JP Selecta, Spain). Samples were finally centrifuged (16,000 ×g, 21°C, 5 min) to precipitate nonsolubilized proteins and volumes of 15 μL were loaded in polyacrylamide gels (12.5%) under denaturing conditions (SDS-PAGE). Prestained Page Ruler (Thermo Fisher Scientific, Madrid, Spain) was used as molecular mass marker. Extracellular proteins were separated according to Laemmli [18 (link)], in an electrophoresis buffer containing SDS (1 g/L), TRIS (3 g/L), and glycine (15 g/L) (Sigma-Aldrich) at pH = 8.7, under a constant current of 40 mA. To visualize proteins, gels were stained with colloidal Coomassie blue (GelCode Blue Safe Protein Stain, Thermo Fisher Scientific), according to the manufacturer's instructions.

To prepare the sample, 30 µg of each recombinant cysteine mutant (EA1_N703C/N340C, EA1_N703C/D343, and EA1_N703C/K372C) were boiled with and without 1 mM of 2-mercaptoetanol. Samples were analyzed by SDS-PAGE, electro-transferred onto a 0.2 μm Nitrocellulose membrane and blocked with 5% (w/v) skimmed milk for 1 h. The membrane was incubated for 1 h at room temperature with Mouse anti Histidine Tag:Alkaline phosphatase-conjugated (BioRad MCA1396A). After washing three times in TBS-Tween buffer (Tris-HCl pH 8 10 mM; NaCl 150 mM; Tween20 0.05% (vol/vol) for 5 min each wash. The membrane was revealed by incubating for 10 min with NBT/BCIP (Roche) and imaged using the GelDoc imager (Biorad). Prestained PageRuler (Thermo Fisher Scientific) was used as a molecular ladder. The uncropped blot is shown in Supplementary Fig. 20.