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Western Blotting Spotting - Spots on Westerns Blots (Oct/20/2004 )

I have been doing westerns for several years now andhave never had any major problems. Recently however they have started to give me trouble. The problem is that when I expose the membrane to our camera there are "spots" on the membrane. These spots are not burned out areas from the ECL but areas were it appears that there is an absence of anything. It is not a problem with the transfer, as per a fast green stain of the membrane looking for total protein. Where the blank spots are on the picture there is plenty of protein on the membrane. The camera is not the issue, since with an autoradiograph showed the exact same thing. That leaves me with the blocking steps and the primary and secondary antibodies. I have tried different primary antibodies and the problem occurs with each one. I have used new secondary and the problem is still there. The only thing that I can think of is possible the room temperature is to low. Right now our room temp is about 69 degrees. Does temperature matter within a few degrees. Has anyone ever experienced this problem where it looks like that no antibodies stuck to the membrane in random spots and even right in the middle of a band. I have a good picture if anyone wants to see. just e-mail me and i will send it along. biggrin.gif

Please Help


The same thing has happened to me and I cannot understand either


Perhaps it is the membrane. Did you buy a new lot?


Are you using the same containers as previously for the blocking and antibody incubation? We experienced some great background problems when we switched to new boxes, and it took quite a while to find out what the problem was. Using the old boxes, everything was fine.

Other critical points that helped my blots are:
- vortex and then spin down the antibodies every time you use them
- when blocking in milk, stir it over night in the cold room to make sure it is completely dissolved
- if you use methanol-containing transfer buffer, rinse the blot with water before blocking


Are there any bubbles between your membrane and the Sarran wrap, that results in blank spots. make sure there are no bubbles there
have fun biggrin.gif


i have read that this is a result of an "aggregated HRP conjugate". mix your Ab or developing reagent (e.g., SAv-HRP) better or even syringe filter the diluted HRP solution.

good luck!