I am facing problems with my western blots like uneven bands of loading control, Smile effect of the bands/Dumbbell shaped bands, and black spots on the film.
For improving gel quality, you can do the following:
1. Do not increase the protein you load for the allowed limit. For e.g. for 10 well comb gel with 30 ul capacity, you should not use more than 60ug total protein.
2. Use fresh APS.
3. Make sure transfer unit is not getting warm during transfer.
4. If you are using stacking gel run first 30-50 minutes @ 60V.
5. Prepare fresh buffers
The FAPP2 blot looks OK to me, although the antibody is picking up some non-specific bands. It could be a bit cleaner, but there don't seem to be huge problems. The GAPDH blot looks quite different, though. Were they from exactly the same lysates diluted with the same amount of loading buffer? were the gels run with the same buffers? When I've had dumbbell - shaped bands, it's been from an incorrect salt concentration, caused by using too much loading/novex buffer for my lysate.
For improving gel quality, you can do the following:
1. Do not increase the protein you load for the allowed limit. For e.g. for 10 well comb gel with 30 ul capacity, you should not use more than 60ug total protein.
2. Use fresh APS.
3. Make sure transfer unit is not getting warm during transfer.
4. If you are using stacking gel run first 30-50 minutes @ 60V.
5. Prepare fresh buffers
In my case, blocking (BSA out, dry milk in; present in all buffers throughout) solved the inspecificity issue.
First, PVDF membrane?
You should block the membrane with methanol (few minutes prior to transfer). In such case, milk (casein) is not necessary to block the unspecific sites.
I recommend you to read: http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf
This could give you some hint about your problem.
Cheers,
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