Trizma hydrochloride solution tris hcl

Soluble Leishmania antigen (SLA) was prepared as previously described.⁶³ Briefly, L. donovani amastigotes from clinical isolates (Kala‐azar Medical Research Center, Muzaffarpur, Bihar, India) were grown in Medium 199, Hanks’ Balanced Salts (M199; Thermo Fisher) until transformed into promastigotes, then cultured. 2–3 × 10⁹ stationary‐phase promastigotes were harvested from culture and centrifuged at 3900 g for 20 min to obtain a parasite pellet, which was washed three times with cold 1 x PBS and resuspended in solution (10 mM Trizma hydrochloride solution (TRIS‐HCl; Sigma‐Aldrich), 1 mM pH 8.0 ethylenediaminetetraacetic acid (EDTA; Amresco, ELITechGroup, Puteaux, France), 1.6 mM phenylmethanesulphonyl fluoride (PMSF; HiMedia, Mumbai, India) and 50 mg mL^‐1 N‐acetyl‐L‐leucyl‐Lleucyl‐ L‐argininal (leupeptin; Amresco)) at a concentration of 2–3 × 10⁹ parasites m⁻¹. The parasite suspension was sonicated 4–5 times for 15 s at 10 Hz and centrifuged at 2000 g for 30 min at 4°C. The lipid layer was removed from the surface of the supernatant, and the remaining solution was ultracentrifuged at 100 000 g for 4 h at 4°C. The supernatant was removed, dialysed against the PBS overnight and stored at –80°C until used. Protein concentration was measured using a Micro BCA Protein Assay Kit (Thermo Fisher), as per the manufacturer’s instructions.