I am trying to detect EGFR in Mouse Embryonic Fibroblast (a transformed cell line) and I am consistently seeing a band near 37KD, and a very faint band at correct mol wt (175KD). Is this some degradation product? Is there anything I can do to prevent this?
I would suggest to try a different antibody (or several different antibodies). If this doesn't help, check if you larger proteins are transferred OK in your western. But in our experiences, bad antibodies are the most common source of incorrect labelling.
Hi,
I am wondering what is your transfer condition?
As you said already this molecule is really huge and this would be a problem in terms of transferring the protein to membrane from the gel. So, either you increase the electricity power by manipulating the volt and amp of your condition or you could increase the time. In the first place, I would strongly recommend you to optimize this conditions in your hand.
Best,
Which antibody are you using? Try using the antibody in the paper and datasheet attached below. Hope it will be useful.
I use transfer buffer with tris-glycine-methanol (Transfer at 85V, 2 hrs). Also tried sodium bicarbonate buffer (Transfer at 300mA, 2 hrs). No difference between the two.
The antibody I am using is from Cell Signaling (Cat. 2232).
I strongly agree with Birger's suggestion that you may use more different sources of antibodies and also pay attention to the cell culture with or without serum (containing growth factors) that may affects phosphorylation of EGFR. Good luck!
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