Hi,
I have unusual western blot. My beta actin antibody is binding actin in ONE part of the western blot and the other one is binding somewhere else. I am not sure what happened. Can someone suggest
Hi
First of all, you have to check protein band distribution by SDS-PAGE with coomasive, or other dyes.
check protein concentrations of your samples.
be carefull during blotting (air bubles)
unfourtunatly, your western blot seems a bit terrible. you need too much optimization
Hi
this western seems to me it need a bit of optimization!! I think your samples are running unequal, something that comes from bad loading or not a constant voltage applied to the system.
Make sure you are using a good running buffer as well
Hello, In your W.B. you might consider following points
As per your western blot image (though image is not clear), you might consider following points
- your protein is not completely resolved, this might be because of voltage fluctuation or some issues with your SDS buffer.
- Please optimize the Methanol concentration in your transfer buffer (you can consider using 20~40% Methanol). Please maintain temperature during transfer.
- Blocking is not proper (uniform) as per your image.
- Did you touch your membrane? Please handle the membrane carefully during transfer. What kind of membrane are you using (PVDF or Nitrocellulose)?
Please mention your complete protocol; we might help you to narrow down your optimization condition for next run.
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