I am working on recombinant protein (Ig's) expressed in form of inclusion bodies. Upon giving certain treatments there is a up shift in protein band. If my protein is degrading during the process; there must be "downshift" but here the scenario is different. I am unable to find out what may be the possible causes? What changes might occured in protein since protein is in the form of IB's.
Image: All lanes have same samples of same batch with different treatments. Lane 9 is old previous ref sample; where protein should be.
Thank You.
Insufficient reduction of disulfide bonds is one possible cause for a protein to run slower than expected on SDS-PAGE. Another is retention of a signal sequence that is normally processed proteolytically.
In this case, though, I wonder whether it is really the original sample that is running faster than expected due to some specific proteolytic cleavage, since most of the samples run at the higher position.
Dear Vikas
Sometime phosphorylation increase negative charge on molecule and should cause a upshifted band in SDS-PAGE. It is demonstrated that phosphorylated protein will run slower and be the upshifted band .
Thank you
Marius
Thank you all for your valuable answers.
Adam Sir, what if I add more amount of reducing agent and or SDS during sample preparation. Will it make all samples to run at same position. Since, I have seen that crude IB's runs longer distance than treated IB's. In the given figure, In lane 1-3, half of protein are upshifted and half are at original position. Whereas, in lane 7&8, all proteins are upshifted.
Your suggestion is certainly worth trying.
Since you haven't mentioned what the "treatments" were, it's impossible to say whether they may have made any difference in the migration of the protein.
I think it would be worth cutting out the bands and submitting them for mass spec to determine the masses.
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