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.