Hi,
I am purifying a membrane protein of 54Kda (with his tag) using DDM Detergent. After Ni NTA chromatography, I can see my protein band but is mostly accompanied by 42 kDa and around 74 kDa band.How can I remove it or know whether these contaminats are helping my protein folding( maybe a chaperone).
Another question is that whether I can perform mass spec of my protein (in gel trypsin digestion) if my protein has been solubilised in detergent??
If you expect the a different oligomerization states of these proteins; then you can try using Gel filtration chromatography. I assume that you have given differnet attempts to get rid of the contaminations in NiNTA like using stringent imidazole washes, altered NaCl concentrations etc.
I think for Mass Spec you need to ppt the protein or get rid of the detergent.
The detergent should not interfere with in-gel digestion and resulting mass spec because (a) the detergent and protein will be separated by the gel electrophoresis, and (b) the gel piece is washed to get rid of SDS prior to digestion, which would also remove any residual DDM.
As previous answer indicated, I would definitely run a size exclusion chromatography step after the IMAC. Your protein is 54 kDa (as a monomer), add to that the DDM, which gives your protein-detergent complex a molecular weight at ca 110 kDa (DDM micelle is ca 50-60 kDa i size). If your protein is expected to oligomerize, the size difference to the other co-purified proteins will be likely even larger (depending on how they oligomerise).
In gel trypsin digestion works fine for membrane proteins. The problem that you might encounter is that the resulting peptides are too hydrophobic to fly properly in the MS, but that depends a bit on what kind of MS you will be using for the analysis.
If you get chaperones co-purifying, as is frequently seen in Ni-NTA chromatography, try a washing step containing ATP.
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