I am using E. Coli B21 as a vector with pGEX-2TK plasmid. I induced my fusion protein with IPTG, creating GST-protein of interest. I loaded an SDS-PAGE gel: in the first well, I loaded total proteins from the bacteria, second well, the induced proteins, and the third well the purified proteins attached to the sepharose-glutathione beads (technically, only GST-protein of interest). In the second and third well, I have bands that correspond to my protein of interest + GST (around 37 kDa), but I also have other bands of smaller weight. I read that my protein of interest could have degraded and left smaller fragments (which migrated farther), but I am not sure I am getting the full story. Is it possible that the induction with IPTG could have transcribed other genes than my fusion protein? It could be, but how would it have bound to the sepharose-glutathione beads then?
This is for a university lab class, so my technical vocabulary might not be the best! If it helps, my protein of interest is amphiphysin IIa (and I am using amino acids 329-425).
It is also possible that the affinity purification was incomplete, meaning that some contaminating proteins came through the purification. A single purification step is usually insufficient to get pure protein unless the level of overexpression is very high.
If this were an actual research project, you might add another chromatography step to the purification method, such as ion exchange or gel filtration. You might also cut out the lower molecular weight bands from the gel and submit them to a facility for mass spectrometry sequencing to find out if they are proteolytic fragments or contaminants. If they turned out to be proteolytic fragments, you would try to make some changes in your methodology to reduce the problem, such as adding a protease inhibitor cocktail to the lysis buffer, being careful to perform all processes at low temperature (4oC), and perhaps switching to a protease-deficient expression strain.
Dhiraj Srivastava
it was also a possibility I had considered, so it is good to receive confirmation, thanks.Adam B Shapiro thank you, that is extremely insightful! I might add that to my conclusion as a source for errors.
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