I've incubated my western-blot membrane with both primary and secondary antibodies and i just realized at the end that i've used a lower concentration of my primary antibody for my loading control than i should.
Can i reincubate this membrane again with the right and higher dilution of this first antibody? or there's any alternative to solve this?
Thanks!
You can try to strip the blot and re-incubate with the primary and secondaries. There are commercially available buffers - https://www.abcam.com/protocols/western-blot-membrane-stripping-for-restaining-protocol
- https://www.thermofisher.com/order/catalog/product/21059#/21059
You can also make your own buffer for the same.
Good luck!
@Sathya
You can actually. In our lab, we usually exposure the membrane and then block it again for 2 hrs and then use another 1st antibody for the same membrane, and still see the very good results. So, you can either actually expose your membrane with substrate and try see the results, and then block it again, and add right concentration of 1st antibody and so on; or just add straight 1st antibody with correct dilution factor and next day do the secondary antibody and expose your membrane.
You can reincubate the membranes to achieve stronger signals. There is no need to strip as long there is no overlap with the other bands already on the membranes.
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