Hei,
Today I got a very strange western Blot. Normally, the antibody is working perfectly but today, the bands are looking very strange- there are shadows around the bands (see attachment). What might happen to these bands?
Your pictures don't show any MW markers. If you see the same shadow bands in your MW markers, the problem is in your gel or buffers. If MW markers look good, the problem is with your samples (proteolytic degradation, variations with your particular experiment conditions, post-translational modifications, differences in glycosylation, and so on).
Your pictures don't show any MW markers. If you see the same shadow bands in your MW markers, the problem is in your gel or buffers. If MW markers look good, the problem is with your samples (proteolytic degradation, variations with your particular experiment conditions, post-translational modifications, differences in glycosylation, and so on).
Hei,
Thank you for your fast reply!
yes, the MW marker is looking similar (see attachment). Thus , there was something with the blot... And in contrast to the other blots, I got many unspecific bands...I read that this may happen when the running/transfer buffer has been used too often. Maybe this is the problem...However, even if the blot is not looking good, it shows perfectly what I have hypothesized. Hopefully the repeat of this experiment will be good so that I have a nice blot for my paper...
How long was the exposure time? You can get this with a strong signal, if you move the film after putting it on. If you're using film that is.
We are using a fluorescence detection method. The "green" picture is actually looking much better. Here, the shadows are not as visible as using black bands.
However, we are always using a picture with black bands for publications and therefore, I hopefully will get a nicer picture when I repeat the experiment. Anyway, I have to repeat the experiment at least one time before I publish. So, there is a good chance to get a better picture. And if not, I may use this picture for my publication as it shows very clearly what I want to show (it`s about PARP cleavage and there is a difference in PARP cleavage where I suspect to have a difference and even if the blot is bad, the differences are clear...).
Yesterday, I added a second antibody against a second protein of interest. As this protein is much smaller than PARP, I did not strip the blot before adding the antibody.
I just analyzed the expression of the second protein and as you can see, the bands from PARP are looking much, much better. Maybe I should wash even more to get even better bands...
Actually, we were using a PARP antibody that was more than 10 yeas old. As the old one was empty, we got a new antibody . The new one should be the same as the old one (same company, same number but who knows if this is really the same antibody...)...Thus, may it be possible that I used too much of the new antibody? That we should dilute more than we are used to?
Were you able to find out what was the reason for those shadow bands?
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