I have purified several his tagged proteins using IMAC. To clean up the samples further I ran them through a GF column (Superdex 200 increase 10/30Gl). When I ran an SDS sample containing the combined elution fractions of a single monomer peak I find additional bands (including the tagged protein).
. Is it worth running SDS samples of each elution within the monomer peak - might one fraction be purer than the others.
. I expect this is a resolution problem - sample viscosity may have been a problem resulting in a fairly broad peak (containing contaminants and degradation products)
. Protease inhibitors were added to cell lysates - didn't seem to affect degradation products.
Any feedback and advice is much appreciated!
It seems like a good idea to check each fraction in the monomer peak to see if there is any fractionation.
You probably need to add an additional chromatography step to get higher purity. Ion exchange would be a good choice.
I think you should work on the IMAC buffers to break the nonspecific protein interaction than to indulge in analyzing the SDS page bands of GFC fractions. This will save your time.
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