I am doing western blot and the problem that I encounter is that the target protein and housekeeping proteins' molecular weight is close enough to cut the gel to probe on separate membranes. However, they get separate enough to distinguish between two easially.
My question is after probing with one ab, do I need to strip before probing with ab for other protein?
Dear Abdul
you don't need to run two gels, to cut the gel or strip the membrane. you have only to do the two antibodies in two different moments. I normally use an anti HA ( rabbit) for my protein and then an anti housekeeping (Mouse) to normalize without any kind of problem. I can ensure that everything will work without problems. I have done 400 wb in the last three years in this way.
regards,
Francesco
you can mix the two antibodies if they have the same secondary conjugate antibody
you can run 2 antibodies on the same gel if they have the same secondary antibody. However if not you should strip the membrane.
and i prefer if you run 2 gels each for each separate antibody
Dear Abdul
you don't need to run two gels, to cut the gel or strip the membrane. you have only to do the two antibodies in two different moments. I normally use an anti HA ( rabbit) for my protein and then an anti housekeeping (Mouse) to normalize without any kind of problem. I can ensure that everything will work without problems. I have done 400 wb in the last three years in this way.
regards,
Francesco
Thomas Schwarz · Medical University of Vienna just published nice way of rapid stripping:
it works quite well on PVDF and NC membranes. I regularly use this to reprobe my western with a-Tubulin antibody as a loading control.
- remove previous Substrate by washing with TBS-Tween
- wash the membrane with ddH2O for 5 min
- wash the membrane with 0.2M NaOH for 5 min
- wash the membrane with ddH2O for 5 min
- go on with blocking and antibody binding
You need not to strip the membrane. You can reprobe with loading control antibody after you visualize your protein of interest. Or as others have suggested, you can use mixture of both the antibodies. I have done this even when the two antibodies are from different species. In this case you have to use mixture of secondary antibodies as well. (Just make sure that your antibodies do not react or affect each other)
Choose the primary antibodies for your two proteins raised in different species ( eg, rat or rabbit against mouse) and incubate with both the antibodies together ( primary) and then secondary. This should work. Usually it is not good idea to mix the primary antibodies raised in same species and never know you might get cross reaction.
You may strip the blot if using the same to avoid cross reactivity, other wise if you have a MW marker lane next to it then just cut the blot horizontally and incubate respective primary antibody in it and you are done. As suggested above, mixing antibodies also works well.
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