protein concentration method - want really high yield (Mar/10/2009 )
Hi. I have to concentrate my protein sample which is really precious.
I don't want to lose any of my protein. It could be denatured.
What would you recommend?
I'm thinking of TCA ppt and ultrafiltration. If you have better idea, please share.
Thank you in advance for your tips of advice.
I would use filter membrane. no TCA precipitation. Some proteins are difficult to resuspend.
I use TCA when I want to do SDS-PAGE (I resuspend the pellet in laemmli buffer)
What do you want to do with your newly concentrated protein? You could precipitate with ammonium sulphate to concentrate, then dialyse to get rid of the amm. SO4.
I don't like to denature my protein and hate concentrators as the protein sticks to the membrane and I loose out a lot. I generally use anion exchange chromatography and concentrate it by using a running gradient. However, in case that doesn't work, I would transfer the protein in a dialysis bag with a cut-off and then hang it in the cold room for the buffer to drain off.
It sounds good to me but have never used it as FPLC works in most of my proteins.
If you use it and it works for you, I would appreciate if you could send me a message.
Ammonium sulphate precipitation is a reversible event as you are not denaturing the protein, just 'salting out', kind of reversible aggregation. It's a standard practice.