Agarose native gel electrophoresis analysis of thermal aggregation controlled by Hofmeister series

The effects of salting-in and salting-out salts defined by Hofmeister series on the solution state of bovine serum albumin (BSA) in 50 mM Tris-HCl buffer at pH 7.4 before and after thermal unfolding at 80 °C for 5 min were examined using agarose native gel electrophoresis and mass photometry. Gel electrophoresis showed that salting-in MgCl2, CaCl2 and NaSCN resulted in formation of intermediate structures of BSA upon heating on native gel, while heating in buffer alone resulted in aggregated bands. Mass photometry showed large loss of monomer and oligomers when heated in this buffer, but retaining these structures in the presence of 1 M MgCl2 and NaSCN. To our surprise, salting-out MgSO4 also showed a similar effect on gel electrophoresis and mass photometry. Salting-out NaCl and (NH4)2SO4 resulted in smearing and aggregated bands, which were supported by mass photometry. Aggregation-suppressive ArgHCl also showed oligomer aggregates upon gel electrophoresis and mass photometry. [Display omitted] •Thermal unfolding of BSA was studied by agarose native gel electrophoresis and mass photometry.•MgCl2 suppressed aggregation of BSA and enhanced intermediate structures.•Aggregated and intermediate species were stained by SYPRO Orange dye.•MgCl2, LiCl, CaCl2, GdnHCl and NaSCN enhanced the intermediates.•NaCl, ArgHCl and (NH4)2SO4 resulted in smearing or aggregated bands and formation of large particles.