Hi
i would like to know because i did not wash off the ponceau stain from the nitrocellulose membrane thoroughly, and as a result the recycled primary antibody solution has ponceau dye in them. Will this affect subsequent primary antibody stainings?
should i just discard these?
Hi,
After ponceau stain you have to wash with TTBS, then saturate with milk, then wash milk thoroughly and to finish add your primary antibody. After all these steps, it seems difficult to have remains of ponceau. Are you sure it is really the ponceau in your antibody ?
We block with serum and do not wash before adding the antibody. I have occasionally got ponceau in my antibody solution. It is a very faint pink tint. I have no problems when I reuse this.
After ponceau staining wash membrane with TBST, and water.
It doesn't affect Ab, you can recycle it.
Hello
Yes i agree with Bhaskar Gouda , you can wash membrane with TBS-T + water
Also you can see links here which discuss the same issues :
https://www.researchgate.net/post/Problems_Stripping_Western_Blots
https://www.researchgate.net/post/Does_Ponceau_S_staining_effect_the_binding_of_the_primary_and_secondary_antibodies_in_western_blot
Good luck
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