This has been a problem with my Westerns lately, I'm pretty sure it's not a transfer issue, but it looks like the secondary antibody is being scraped off somehow? I'm not sure how this could be happening, if anyone has any idea please let me know.
Hello. In a lot of of the problem areas it certainty looks like small air bubbles got between the gel and the membrane during the transfer which prevented the protein from transferring in those spots.
In the areas where it looks like slashes are appearing it could be that the surface of the membrane you are transferring onto has been damaged at some point. It might not even be visible to the eye.
I would try brand new PVDF or Nitrocellulose and be more thorough using a roller during the transfer to also avoid air bubbles and see if this improves the results.
I also like to let my gels sit in transfer buffer for 5-10 mins to ensure all the SDS is out as this creates a high chance of bubbles.
I agree with Evan Kerek about bubbles between you gel and membrane, and also about using the roller and closing the "sandwich" really tight. Also, you should be careful to make sure all buffers are completely clear and when wetting the membrane, make sure it stays smooth.
It looks a lot like uneven pressure in the transfer case to me, with the top of the get being resolved well, but the bottom becomes disturbed. I agree with other respondents to use the roller well and ensure proper closure of the transfer sandwich.
In order to confirm your transfer efficacy, you can use the Coomassie Blue or Ponceau Red total protein staining after the transfer, but before blocking the membrane. This will allow you to distinguish the problems associated with the gel run and transfer and any issue your membrane might have it will also allow you to certain degree ascertain how even is your protein loading and scrap clearly unsuccessful runs, without wasting time and antibody on incubation. In a separate container, you can incubate the gel as well, if you see some bands or areas with bands remaining on the gel that will be an indication of transfer issues. While unlikely it is also possible that your membrane might scratch against the container that you use for the incubation (there must be some irregularities on the bottom of the container. This way total protein staining will indicate clean membrane transfer but "scratches" may appear after primary incubation.
Thanks everyone for the replies. I think the problem came from me using an aspirator to remove the secondary solution instead of dumping it. The force of the vacuum line was likely tearing off the secondary. I have stopped doing that for my more recent Westerns and they are looking fine.
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