I had been having beautiful blot transfers, but in the last few days I have ran into issues where there is high background staining and poor band resolution. The background staining is very mottled/grainy looking, similar to the pattern on the paper that divides the nitrocellulose membranes in the packaging. I cannot think of anything I have done differently procedurally to get bad transfers. I tried troubleshooting various aspects. I tried hand cast vs. precast gels, same result. I tried samples that had previously not given me issues, came out mottled. I tried a new box of membranes thinking that maybe there was something wrong with the box, same results. Granted, the boxes of membranes all have the same lot number, so they could all be bad?
I'm really at a loss for what to do and why they are like this. Any suggestions would be greatly appreciated. Thanks!
Have you tried staining the gel with Coomassie to see if proteins are separated nicely? This might not be a problem of bad transfer but bad PAGE.
When I had a problem with transfer problem was methanol that we used for transfer. If you changed manufacturer lately try funding the one used previously and use it.
Hey folks! so issue partially solved! I came across an article discussing troubleshooting and it said that if you haven't changed anything in your protocol and things have started looking funny that it's most likely your scotch brites have gotten thin or contaminated and that they should be replaced every month if they are in heavy use. A temporary suggested solution was to double up on the filter paper in the transfer sandwich, and it worked! No more mottled background staining!
A separate issue that we have noticed in our lab however is a band appearing in all the lanes, including the lanes with only laemmli buffer loaded. It only shows up during ponceau staining and doesn't appear to interfere with chemiluminescence but we aren't sure what exactly is causing it. We use 2X laemmli buffer from BioRad mixed with betamercaptaethanol 30:70 ratio (BME to Lam.). Does anyone know what this might be? (seen in the image below at the upper orange ladder band gel-wide)
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