We are working with a protein that is expressed in inclusion bodies. We are using 4 M urea to solubilize, however when we start the refolding using dialysis and decreasing concentrations of urea, we are losing a lot of protein by precipitation. Someone can suggest another methodology to solubilize and refold the protein?
Thanks a lot,
Fernanda
Well there is no single protocol that works for all proteins. In general you need to have a buffer where protein precipitation is reduced, for this you need to have a charged protein molecule, a reduced protein concentration, a lower temperature, etc.
Hi Fernanda, I have had some success in refolding proteins by incubating them with 2M arginine (pH 8) with 5 mM TCEP for 1 hour at RT and dialyzing.
@Steingrimur Stefansson, thanks for your answer. I have a question about your protocol. Do you use urea to solubilize?