Basically, when I load the same volume and sample into each well (15 wells), only the first half the lanes result in an accurate transfer, whereas the second half do not. This is clearly seen after film development.
I have mostly brand new Biorad western blot equipment.
I don't think it is a pH or buffer issue because then ALL the lanes would have issues, but this is not the case.
Perhaps it is a sample + sample buffer mixing issue?
Something wrong with the power box? i.e. the current isn't evenly distributed?
Any help would be greatly appreciated!
Is the system based on tank blotting or semi-dry method?
I am working with semi-dry method from Bio-Rad, with the old semi-dry transfer cell which should work for up to 4 blots in parallel - in our lab they work fine for 1-2 gels, but as soon as you have 3 or 4 gels, only 2 gels will look good, the other one will be either not working at all (in case of 4 gels in one blotting unit) or only half of the protein will be transferred (can't remember if it was left to right or up to down). Therefore, I am not blotting more than 2 gels in one blotting cell.
You can do some Ponceau staining of your membrane after blotting. This will show the overall protein that you have on the membrane. Film development is in my opinion a too late step for searching of the problem.
May be your western blot apparatus is not equally distributing the power. To check, you can try to keep the first half of your lanes in a position where your blot doesn't show any transfer (as you said).
Secondly, try to do ponceau staining before proceeding with immunoblotting. It will safe your time and energy.
Good Luck
I agree with above suggestion. And would like to add that If a PVDF membrane was used and not entirely sunk in the methanol ( for example), may cause problem. Also, make sure there is no an air bubbles between a gel and a membrane.
Good luck
Thank you everyone! I am currently testing different apparatuses and ponceau staining. Hopefully everything works from here on out.
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