Freezing eluted His tag protein - (Jun/03/2010 )
Hi,
I am purifying a His tag protein from cell media via an IMAC column in an elution buffer containing 200mM imidazole, is it ok to freeze the protein in this elution buffer as I have heard it could damage my protein. If this is not ok how do I go about doing a buffer exchange? I have some columns from Millipore that I have used in the past but I have now been told that the imidazole in the buffer could cause the protein to stick to the membrane and I wont be able to wash it off.
Any advice anyone could give me would be great, thanks.
BB25 on Jun 3 2010, 01:26 AM said:
I am purifying a His tag protein from cell media via an IMAC column in an elution buffer containing 200mM imidazole, is it ok to freeze the protein in this elution buffer as I have heard it could damage my protein. If this is not ok how do I go about doing a buffer exchange? I have some columns from Millipore that I have used in the past but I have now been told that the imidazole in the buffer could cause the protein to stick to the membrane and I wont be able to wash it off.
Any advice anyone could give me would be great, thanks.
Millipore columns works in this situation. I have purified a tagged protein in an elution buffer containing >200mM imidazole. After ultrafilter most of my protein was recovered. imidazole buffer maybe cause the protein to stick to the membrane but that should be a little.
I dialyse my protein after elution in250mM imodazole. I use Dialysis Cassettes from Pierce!
Greetings Franzi!
Benita on Mon Jun 7 11:39:42 2010 said:
I dialyse my protein after elution in250mM imodazole. I use Dialysis Cassettes from Pierce!
Greetings Franzi!
Maybe you shouldn't freeze it in this buffer, we use a special freezing buffer. I think thawing and freezing could also damage your protein.
I was working with 2 different enzymes, both eluted with 250mM imidazole. After one freezing-thawing cycle, one of the enzymes lost the activity, whereas the other one retained it to the same extent. So, I think it varies greatly from protein to protein; obviously, freezing it not advisable.