I am running a EGFR western blot, and despite the protein appears quite specific is not in the correct size (close to 180-200) while should be around 130 KDa.
I am not so sure what is going on, if I am detecting an unspecific protein (although band is intense), not sufficiently denaturalized or is not entering correctly in the gel (7.5% PAGE gel).
Any one would be able to provide me with a possible way to troubleshoot?
Hi Carmen,
Are you running purified protein or a cell lysate? If it is a cell lysate, it could be that your EGFR is highly glycosilated increasing its apparent molecular size. Try pre-deglycosilating your samples:
https://www.neb.com/applications/glycobiology-and-proteomics/removal-of-n-linked-and-o-linked-glycans-from-glycoproteins
Best,
thank you. indeed seems a problem not in the extraction, instead in the denaturalizing the protein which seems not effective with the b-mercaptoetanol, as remain not entering in the separating gel.
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running