Dear All,
I google many guides and they suggest that larger proteins are more difficult and take more time to transfer from gel to membrane in western blot.
Is it possible that some proteins, which has lower MW (molecular weight), might transfer to membrane more difficult/slower than larger MW proteins. For example, transfer at the same condition from the same gel to the same membrane,then assume that the sample has both proteins, and the sample doesn't run out off gel. When I developed my bands only larger MW protein signals appeared, not lower MW proteins. Why?
Thank you very much
Cheerio,
Provided you run SDS-PAGE (not native), smaller proteins should be faster during blotting. Try to stain the gel after blotting to see, whether your smaller protein is still there (or it just was not trapped by the membrane).
Martin
It is likely that your smaller protein does migrate faster... So fast that it has passed through the membrane and out the other side and is gone. You could try shorter times to see if that improves your signal.
Based on the molecular weight ladder and the protein size of your interest select the correct percentage SDS-PAGE to run. After transfer stain your membrane with coomassie brilliant blue or any other stain such as Ponceau S. If everything looks alright then only proceed further. There might be a problem in your transfer buffer. Check for composition, pH of your transfer buffer.
Alternatively, you can try blotting with two membranes at once - one PVDF, the other nitrocellulose. When your protein of interest runs out of the gel and is not retained by nitrocellulose, it might be trapped on PVDF or vice versa.
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