Hi,
Do we need stripping for western blot membrane(PVDF) when we have to re-probe with a primary antibody having different origin in comparison to the previous one. Previously I had probed with p-tau(rabbit) having strong signal. Now I want to re-probe with total tau(mouse) with same molecular weight.
Thank you very much.........
Hi,
Basically once you develope the membrane u will not be able to tell which antibody has detected the band. It is albeit safer to strip. Stripping is extremely easy.
Actually if your secondary antibodies are species-specific (some aren't), then you can just destroy the HRP activity on your rabbit secondary with sodium azide (.05% in PBS). Then incubate with your murine primary antibody and visualize with your anti-mouse secondary as you would normally.
Hi there,
In your situation (probing roughly the same object with 2 different systems) stripping is absolutely necessary in order to make sure the signal you measure for the second probing is actually mediated via the binding of the primary antibody of the second probing with no contribution due to a possible crossreaction between primary and secondary antibodies or even persistence of the protein/antibody complex formed during the first probing...
Its better to use stripping solution supplied by Thermo scientific. Don't take a risk to re incubate it with other antibodies. That may give you some non specifec bands.
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