How many antibodies (of different molecular weights to distinguish from one another) is it ok to run on a particular gel during western blotting? I ran GFAP, GAPDH, and BDNF all on one blot as they have distinct weights that shouldn't be near one another, with 5% BSA blocking agent. I got a very thick band for GFAP at 48kDa (below what was expected based on the Abcam data sheet, and 2 smaller bands which I believe are derived from it? Then the second band from the bottom is GAPDH and the last one is BDNF.
Would this image be ok to use for analysis?
I think it would be a good idea to do a blot with each antibody separately to verify that each produces a band that will not overlap with the band of another antibody. Then you can multiplex them.
Hi Stephen,
the most accurate would of course be 3 different blots, but if you not have enough material maybe horizontal cutting of the membrane at appropiate sites could be a solution. If you apply marker on the first and last lane, you could carefully cut horizontally at the right molecular weight between the 2 marker lanes (or 3 if you choose to apply a marker lane in the middle). I have always block the membrane first, than cut and then block again (but this is maybe too cautious). For each of the stripes you can then use the suitable antibody.
best,
Carsten
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