Concentrating recombinant protein without dialysis - (Jan/02/2008 )
Hello,
I am expressing a recombinant protein in E. coli that is HIS tagged for generating antisera in mice. I can purify the protein, albeit in low amounts, but when I put it in a dialysis bag to get rid of the 8M urea and then on PEG to concentrate it, I lose all my protein. We've tracked the problem down and it seems to be sticking to the dialysis bag. Now we are looking for some way to get rid of the 8M urea and hopefully concentrate the protein so that we can use it to immunize an animal. Does anyone have a way to precipitate the protein out of urea? I am familiar with TCA and acetone precipitation but I don't know if that will precipitate the urea as well. Any suggestions?
Bettina
tca will not precipitate urea but will denature the protein. acetone probably won't precipitate the urea at the concentration that you will need to precipitate the protein.
when you dialyze, do you include salt in the dialysis medium? it helps prevent binding to the membrane.
you could use a centricon or amicon to concentrate the protein (and to reduce the urea concentration by diafiltration).
when you dialyze, do you include salt in the dialysis medium? it helps prevent binding to the membrane.
you could use a centricon or amicon to concentrate the protein (and to reduce the urea concentration by diafiltration).
you may use dialysis such as slide-a-lyzer (Pierce) or as mdfenko suggests spin filtration units f.i. from Pierce with low protein binding capacity;
I think centricon or amicon which was integrated to Millipore are not more provided; they offer now alternative products...
I am actually having a similar problem with losing my protein upon buffer exchange/concentration with dialysis tubing, microcon and centricon's. It seems to aggregate and stick to all membranes. I haven't come up with a better solution, but if anyone has any ideas, I would love to hear them. I really need this protein.
We are actually just shooting the washed beads with the protein still bound into the mouse to make antibody. I am going to try acetone and TCA precipitating it to get rid of the urea and I can let you know how that goes if you want.
you may need to increase the ionic strength of your buffer to prevent binding to the membranes.