5 replies to this topic

[BB25]

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.

[123zhangxu]

BB25, on Jun 3 2010, 01:26 AM, said:

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.

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.

[Benita]

I dialyse my protein after elution in250mM imodazole. I use Dialysis Cassettes from Pierce!

Greetings Franzi!

[shilpeejnc]

[quote name='Benita' timestamp='1275910782' post='74271']
I dialyse my protein after elution in250mM imodazole. I use Dialysis Cassettes from Pierce!

Greetings Franzi!
[/qAfter thawing the frozen elute With 500mM imidazole I gaot pecipitate.And after passing through centricon I recoverd very less protein

[Benita]

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.

[ram]

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.