Dear all!!! I am purifying a protein of 30 kDa MW. I purify through Ni-NTA agarose - anion exchange chromatography and finally with size exclusion chromatography. After the final purification, I am getting an extra band of 23 kDa MW in SDS PAGE. But when I do western blotting, only the 30 KDa protein shows the band. Kindly suggest me to get rid of that 23 kDa protein?
The 23 kDa band could be the proteolytic cleavage product of your protein. Is your antibody polyclonal or monoclonal? It is possible that your antibody is raised for a region which is missing in the cleaved fragment.
The 23 kDa band could be the proteolytic cleavage product of your protein. Is your antibody polyclonal or monoclonal? It is possible that your antibody is raised for a region which is missing in the cleaved fragment.
In line with Richa's response, you could perform an in-gel digestion of the 23kDa band and analyse it by MS. If tryptic fragments match your target protein, then it must be a cleaved product. You could then retrace your steps to better understand where the truncation could have taken place.
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