Hello, I am currently expressing a GPCR in sf9 insect cells. I transfected them and performed a protein expression test (western blot) using both an anti-His and anti-GPCR specific antibody. Both produced bands at the expected size but there are other bands above in both cases as well? Any advice to reduce theses would be great as I am trying to get the maximum expression possible.
Thank you!
GPCR are known for post-translational modification such as glycosylation. I had the same thing as you with receptor kinase, after deglycosylation (using a kit from NEB) clear single band! I cannot say for sure it is glycosylation however, it could be several other types of post-translational modifications. Rule out phosphorylation since it generally generates very small shift.
Hi,
I assume your samples were run under reducing conditions?
Could also be protein aggregates; try heating your samples for 10 mins at 70 C prior to loading, rather than boiling.
I don't know what your markers are but to me it looks like dimers and trimers of your protein (more so on first blot). Make sure that your SDS loading buffer is fresh (and has fresh reducing agent). Also if you are using precast gels make sure to add the recommended antioxidant to the top buffer chamber.
I agree with both Hugo and Nick. GPCR are known to behave wildly in western blot experiments. I've seen some WBs of GPCRs overexpressed in 293T cells and it looked like a terrible mess. These proteins harbor a lot of post-translational modifications that can result in these shifts. If you want to be sure that the higher MW bands are really your GPCR with PTMs, you could always try to make an IP of your protein, run it through a SDS PAGE, stain it, cut up the band(s) and analyze them by mass spectrometry.
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