Renaturing Protein after Acid Elution - (Mar/14/2007 )
Hi all...
I am eluting a protein from an IP using [100mM] Glycine pH 3.0. Now, in order to renature the protein after elution, should I raise the pH slowly, say over 3 hrs, OR raise the pH quickly/immediately to reduce the amount of time the protein is in acidic conditions?
There has been some debate in my lab over the best way to do this, so any input would be appreciated.
Thanks.
I am eluting a protein from an IP using [100mM] Glycine pH 3.0. Now, in order to renature the protein after elution, should I raise the pH slowly, say over 3 hrs, OR raise the pH quickly/immediately to reduce the amount of time the protein is in acidic conditions?
There has been some debate in my lab over the best way to do this, so any input would be appreciated.
Thanks.
I think that the better approach is to collect your protein in high-capacity buffer like tris to achieve near neutral pH. (but it depends on your protein mainly) For ex as for antibodies after glycine elution ( add 20-40mkl of 1M Tris buffer pH 9.0 to 2ml fraction size).
Alright, I'll try it that way then. Thank you.
Some corrections: use 60-200ul 1M Tris pH 9.0 per ml fraction
What's the pI of your protein? Assuming it's well away from your proposed new pH, there should not be a problem doing the change quickly.
Do you have much of the protein? If so, try the experiment. Change a small amount slowly, a small amount quickly, see what happens.
Don't be afraid of doing a small-scale experiment. Like one of the signatures here says, every experiment proves something. If it doesn't prove what you want, it proves something else.