Since a few weeks, I don't get any regular bands in the western blot any more. Instead, it looks more like it is broken several times. We first suggested, the probes could have been ruined. I tried it now with some other probes but still had the same problem. Any suggestions what could be the reason?
Many possibilities:
• Protein overload
• All Buffers pH, including Laemmli buffer
I recommend you to make fresh new buffers. Always make 50 mL solutions, because when it goes wrong, you throw all away and make new ones.
Hello,
Many possibilities as Ivan suggested at the same time, you did not mentioned how you did the WB for example. which buffer or loading dye you used? what are the gel running parameters? and so on..
Was the running buffer (and hence the gel) heated up while running the gel?
At this point, I would suggest to provide more information about your WB experiment.
Please do check the loading dye composition and make all fresh buffers and also check the pH.
Best
Pranav
Dear Franziska
High protein concentrations can result in diffuse protein bands after blot development. Reduce the amount of protein initially loaded.
In the addition to aforementioned recommendations, I could suggest you to clean electrodes in your phoresis chamber.
For this blot I used plasma probes so the protein overload is due to the high amount of albumin. My problem is seen the best at the part of the membrane number 3. I already suggested that the buffers could cause such a kind of problems but as the other people here in our lab use the same buffers without having these "broken" bands, I suggested there must be an other reason.
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