Urea effect on mice spleen cells - (Nov/23/2014 )

Hi,

I am going to do lymphocyte proliferation assay using mouse spleen cells in order to assess the cytokine profile of the cells (IFN-gamma, IL-2 and IL-10) after induction with the target protein. I have purified my recombinant protein of interest using denaturing method by 6M Urea. Since my protein disappears after dialysis, I wondered if I could use the protein for inducing cells without dialysis. I would like to know if urea has any negative effect on spleen cells' growth?

Thanks,

can't say for certain if urea will have a negative effect on the cells (probably will) but the protein will be affected and may not give you correct results (if nothing else,the protein will not be folded properly).

if the native protein is soluble then you should perform a stepwise dialysis to remove urea and refold the protein.

mdfenko on Mon Nov 24 14:06:16 2014 said:

can't say for certain if urea will have a negative effect on the cells (probably will) but the protein will be affected and may not give you correct results (if nothing else,the protein will not be folded properly).

 

if the native protein is soluble then you should perform a stepwise dialysis to remove urea and refold the protein.

Thanks for your tips. So, I have to perform the stepwise dialysis

it would be a good idea if you want properly folded protein. keep in mind that you probably won't recover all of the protein.