Gel electrophoresis, recombinant protein, expression in bacteria, molecular biology
Hi,
Because your antibody used for WB recognizes another (variant of the) protein than you primarily seem to observe with SDS-PAGE?
Did you use Coomassie staining, or something else, to visualize proteins in-gel?
Yes I used Coomassie staining for the gel. I sent the incised gel for mass spectrometry, I am waiting for results. But I am wondering why bands on WB would be higher? The protein is 30kDa
Hmmm. Your protein is rather small, perhaps a different ladder will allow for a better estimate of the size? Maybe you could stain your membrane after the transfer to see if it is moving onto the membrane at the expected size too. The Ponceau S stain will show all proteins on the membrane and then can be washed away in the first blocking step. You can also see if there are any bubbles in the bands, distortions, etc. before going through the time investment of Western blotting.
Well it's not just my protein at this point. My supervisor gave me another protein to work with, and the band on the WB appear higher with both our proteins
hmm, that sounds like a quirk in the methods or reagents. Might be time to make new protein loading buffer.
Your gel should have the exact same layout as your Western blot, because your Western blot is a transfer of your gel directly onto the membrane. You might not be noticing the right band on your gel. Maybe the band is very thin by eye with coomassie, or even barely visible, but it jumps out as a very major band with the antibody detection. This happens frequently. Most proteins studied are not the major protein expressed in the cell.
Do you have a different molecular weight ladder for Western and Coomassie stain? If you run the same ladder on both, that may clarify the situation.
Proteins do not necessarily run on the SDS PAGE gel according to their expected molecular weight from the protein sequence. There are several possible reasons for this, such as glycosylation and shape.
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