Hi, so most of my protein that I wanted to express by E.coli BL21(DE3) is in inclusion bodies and hence I used 8M of urea in my binding and elution buffer when I purified the protein with Histrap column.
My question is :
1. what's the bad consequences of using urea? I sure there must be some big disadvantages, otherwise people would just include urea in all buffers without testing the protein solubilization prior. Is it because it's a denaturant hence the protein will be in denatured form? And what will it affect?
2. Will my protein in elution buffer containing urea affect the western blot results later?
3. And to eliminate urea from protein, why do people do dialysis in descending order or urea instead directly in buffer without urea at all?
I appreciate your help. Thanks in advance.