I cut the membrane using ladder guide to two pieces when testing beta actin plus another protein with different m wt and add antibody to each one. If I need to test many proteins of similar m wt, how can I add different primary antibodies?.
Hi Heba,
the way we do it is by using the LI-COR system; we use primaries from different species and secondaries tagged with different colors. In our case one protein band is red and the other one is green. If you are using ECL method is much more complicated. The best way I know would be to use one of the antibodies, develop and then to strip the membrane and reprobe for the second protein. There are commercial stripping solutions but you can easily prepare it. In my experience this works much better in PVDF than nitrocellulose membranes. Another option would be to prepare gels with the appropriate concentration (or gradient) to separate your proteins of interest better, this might be tricky but you could use big gels to increase the resolution power. You could also do 2D gels, separating first according to the isoelectric point of the proteins and then by size. This way the chances of proteins overlapping in the gel are minimized. Finally the obvious answer would be to just run your samples in more than one gel and probe each membrane with a different antidoby.
Hope this helps,
Ezequiel
Reba, if all else fails, you may need to run separate gels or cut your membrane into vertical strips and probe each strip with a different primary antibody (using the appropriate gel loading, of course). Also note, if you are going to try to strip and reprobe a blot, you should test to make sure the stripping worked. Some antibody complexes may persist and give you a misleading response during the reprove. Stripping may also remove or deplete some proteins, that were present in your samples, from the membrane.
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