Protein precipitation problems
Farasha Almadani
Saturday, 18 August 2007 16:46 UTC
Hi everybody I have been purifying a His tagged protein of 19 KD using IMAC under denaturing conditions followed by refolding by Dialysis .My problem is that after dialysis in 1M urea and Tris Hcl the protein has precipitated .Any ideas to overcome this problem
Thanks
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Replies
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This is a well known and common problem and probably related to the way the protein is folding back.
Very important for this to work is the medium in which you’re dialysing. Different pH values may help, slow dialysis, gradually lowering the Urea concentration for better folding… things like that. It doesn’t always work, a bit of luck is needed as always.
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check your protein has any cystein residues, if it has please add a reducing agent such as beta ME in your dialysing buffer and slow stepwise dialysis might hlep you.
good luck… -
Thanks for all who replied ..sorry for being late. I am still working on the same protein and I tried most of your suggestions and it did not work ..I am planning to express it in different media and different hosts …hope it will work
It is a very sad thing that nowadays there is so little useless information.
– Oscar Wilde -
you can try adding different concentrations of L Arginine (100-1000mM)to reduce the precipitation.
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