Problem with protein refolding - protein precipitate all the time !!! why (Aug/16/2006 )

Hi there,

I was able to purify my protien from inclusion bodies under the denaturing conditions using Ni column. I used guanidine as well as urea to denature.
I found my protein in coomasie as well as in wb.
my problem is that when I try to refold the protein after purification (dialysis) but once I go beyond 2M urea or guanidine my protein starts to precipitate !!!!!

I don't know what to do ????

anyone has this problem before? or has any idea??

Thanks

does all your protein precipitate? i generally get some amount of precipitation but a good deal actually remains in solution- you can check this on a gel.
also i think its because urea occupies a huge volume of the buffer, and once it is dialysed out the volume inside my dialysis bag drops to close to 2/3 of its original (prior to dialysis). i assume the pptation occurs in part because of the lowered volume ; maybe there isnt enough volume to accomodate all the protein and also mainly because you will always get some amount of misfolded and insoluble protein ; not all your protein will refold correctly into a soluble form. you can try to improve yields but i think its pretty difficult to get 100 % refolded and soluble protein.

What is in your dialysis solution? Do you have some salt? What kind of protein are you working on - membrane, cytosolic etc?

If you get to ~2M and everything is OK, you could try a rapid dilution into the final buffer, by drizzling the dialysate into a large volume of buffer which is being stirred well. Then purify by a different technique for purity.