u/hana-maki

hello, i'm working with a novel fusion protein. it's highly aggregation-prone, and right now it's in 8M urea-containing buffer. When diluting it stepwise from 8-->4-->2M, the protein remains mostly soluble. however, i'm having issues maintaining solubility when dropping from 2 to 1M. i'm diluting my buffers very slowly (over the course of an hour with constant mixing in the cold), but the proteins still are crashing out. My buffer components are: 50mM tris-HCL, 100mM KCl, 10% glycerol, pH 8 (PI of protein is 9.7). i've tried adding arginine and increasing the salt concentration, but my proteins still crash out. if anyone could help, that would be greatly appreciated. thank you!