emily
Nov10-04, 01:54 PM
Hi everyone,
I am making a protein (20kDa, pI ~5.5) in mammalian cells and then am purifying it by running it over an antibody column, eluting with glycine pH2.5 and neutralizing with Na2HPO4. The protein comes off but I find that there is still what seems to be a lot of my protein still on the column (I boil an aliquot of the affinity column matrix in SDS loading buffer). Is this normal? Can you not usually get everything off the column. Are there any tricks for getting more off the column?
Thanks for any advice!
Emily
I am making a protein (20kDa, pI ~5.5) in mammalian cells and then am purifying it by running it over an antibody column, eluting with glycine pH2.5 and neutralizing with Na2HPO4. The protein comes off but I find that there is still what seems to be a lot of my protein still on the column (I boil an aliquot of the affinity column matrix in SDS loading buffer). Is this normal? Can you not usually get everything off the column. Are there any tricks for getting more off the column?
Thanks for any advice!
Emily