We synthesized some polyHis tagged protein in E.coli and purified with cobalt beads. Its molecular weight is about 65KDa. But we don't know whether it forms oligomers. The coomassie blue stain of SDS PAGE looks pretty clean with some minor contaminating bands with much smaller molecular weights. Mass spec showed some E.coli protein contamination. Is there a quick way that I can use to polish my protein without significant investment? Thanks.
The answer depends on what your contaminants are. IEX or gel filtration are options.
Hi there,
If wondering about homogeneity of the prep, go for SEC to try to separate oligomers/agregates. As the criteria is native MW then you may also get rid of the small size contaminants.
This is a difficult question to answer and would more than likely involve a little trouble-shooting on your behalf. We usually set up a number of small scale experiments using ~50 microL or each resin at a number of pH's. Mix with sample. spin and check supernatant for loss/presence of proteins.
This may involve IEX (This depends on the pI of your protein). Most E. coli proteins (Not all) have pI points in the acidic region so if your protein has a more basic pI it may be possible to get a good clean up using an suitable cation exchanger at higher pH. Even revisiting IMAC: using a less stringent metal ion such as zinc (binds Hist weaker than Cobalt) or even change the buffer, using Tris buffer lowers the affinity of Hist to cobalt resins, what this may mean essentially is the contaminating protein will have reduced affinity for the resin compared to your protein. You could also increase the stringency i.e add increased imidazole in the loading buffer. Carry out a step or gradient elution if not already done so.
- Badges
- Science method