HomeFAQsWhen I run my purified fusion protein on SDS-PAGE, why do I see multiple bands instead of a single band of the expected MW?
FAQ: When I run my purified fusion protein on SDS-PAGE, why do I see multiple bands instead of a single band of the expected MW?
There are two likely explanations for this result. The first is that the fusion protein is unstable, which most often leads to degradation in vivo. In this case, one would expect to see bands between the size of MBP (42.5 kDa) and the size expected for the full-length fusion, since fragments smaller than MBP would not bind to the affinity column. An exception would be if the fusion protein breaks down at the junction between MBP and the protein of interest, and the protein of interest oligomerizes. In this situation, the protein of interest may bind to the fusion protein, and therefore a band the size of the protein of interest can appear even if it is smaller than MBP. The second explanation is that the protein of interest is binding non-specifically to other E.coli proteins, e.g. it has a surface that binds other proteins by electrostatic or hydrophobic interactions. In this case, modifications to the column buffer can sometimes be used to help wash the interacting proteins away. Electrostatic interactions can be weakened by including up to 1 M NaCl in the column buffer, and hydrophobic interactions can be weakened by lowering the salt to 25-50 mM NaCl and including 5% ethanol or acetonitrile in the column buffer. Non-ionic detergents can also be used to weaken hydrophobic interactions, but they can interfere with the affinity of certain fusion proteins.
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