Rapid gold bca protein assay

Approximately 1,000 young adult worms were broken by sonication in 50% Tris‐sodium dodecyl sulfate buffer (25 mM Tris, 250 mM NaCl, 5% sodium dodecyl sulfate, pH 7.4), and the debris was pelleted by centrifugation for 5 min at 20,000 rcf. To precipitate proteins in the supernatant, trichloroacetic acid (TCA, final concentration 9.3%) was added to the supernatant and incubated at room temperature for 1 h. The supernatant was removed, leaving the protein pellet intact. Pellet was washed twice with 200 μl of cold acetone and centrifuged at 14,000 rpm for 5 min. Next, the pellet was dried by placing the tube in a 95°C heat block for 5–10 min to evaporate the acetone. The protein precipitate (TCA‐insoluble fraction) was dissolved in 150 μl of 350 mM NaOH for 1 h at room temperature. Total protein concentration was determined using the Rapid Gold BCA Protein Assay (Thermo Scientific).

Bladder tissue samples were collected at the experimental endpoint for cytokine analysis. The bladder was bisected along the transverse plane, and the upper dome of the bladder was flash frozen in liquid nitrogen and then stored at −80 °C. Protein concentration was determined via bicinchoninic acid assay (BCA), according to the manufacturer’s instructions (Rapid Gold BCA, Protein Assay, Thermo Fisher Scientific, Waltham, MA, USA). A custom-designed mouse cytokine 10-plex kit (R&D Systems, Minneapolis, MN, USA) was used to determine the concentrations of the following cytokines in tissue homogenates: intercellular adhesion molecule 1 (ICAM-1), interleukin 1 (IL-1), interleukin 10 (IL-10), interleukin-6 (IL-6), chemokine CXC ligand 1 (CXCL1), chemokine CXC ligand 2 (CXCL2), P-selectin, interferon-gamma (IFN-γ), LIX recombinant mouse CXC motif chemokine 5 (LIX), and tumor necrosis factor alpha (TNF-α). Samples were prepared according to the manufacturer’s protocol and read using a Bio-Plex 200 Analyzer with Bio-Plex Manager software (Bio-Rad, Mississauga, ON, Canada).

The Mtb Alr enzyme was expressed as the short variant characterized by Strych et al. that features a 24-residue N-terminal truncation relative to the annotated Rv3423c gene^{23 (link)}. The enzyme was His-tagged and expressed in the TESEC Alr- Host strain, which has no other source of Alr activity.
The TESEC Alr Purification strain was grown to saturation in 500 mL of LB supplemented with ampicillin, kanamycin, D-alanine, and 100 mM L-arabinose. A cell pellet of approximately 2 grams was harvested by centrifugation, cooled to 4 °C and lysed with 10 mL B-PER reagent (ThermoFisher 78248) and a standard EDTA-free protease inhibitor cocktail (Merck 11873580001).
The lysate was cleared by centrifugation and passed over 3 mL HisPur spin columns (ThermoFisher 88226). Elution fractions were collected with increasing concentrations of imidazole (Sigma I2399) and checked for the presence of a single band using SDS-PAGE gels (Bio-Rad 4561081) and Coomassie staining. Successful elutions were combined and dialyzed (ThermoFisher 66380) against 20 mM Tris, 100 mM NaCl pH 8.0 for 72 h at 4 °C. Finally, samples were concentrated by centrifugal filtration in Amicon Ultra columns (Merck UFC9010). Total protein concentrations were determined using the Rapid Gold BCA Protein Assay (ThermoFisher A53226) calibrated to a standard curve following the manufacturer’s protocol.