Hi everyone
If i stain my NC membrane with Ponceau Stain prior to probing with antibody of interest, would it interfere with the antibody binding and the subsequent development of the membrane for the signal? A backup reference on this subject would be an add on benefit.
Thank You
Hi there,
Ponceau staining of the membrane is fully reversible during the blocking step prior probing and therefore has no effect on the binding of primary antibody onto its epitope. As mentioned in the article below, Ponceau staing may be use as an alternative for loading control for WB:
Article Reversible Ponceau staining as a loading control alternative...
Hi Vikrant,
We do that all the time.
1) After transfer, wash the membrane with PBS-T or water.
2) Add Ponceau enough to wet the membrane.
3) Protein greater then ~5 microgram and more are seen easily.
Staining takes as short as 2min.
4) Remove Ponceau ( we re-use), wash with water for 1 min.
5) You should see protein bands.
6) If you wash for 30min with PBS-T or 5% milk TBS-T ponceau goes away.
7) Proceed with Primary Antibody
Good luck !
Anand
Once you have the Ponceau S stained membrane, remember to image it (so that you have a reference for where the protein bands are), then make sure the membrane is washed fully to remove the dye before adding primary antibody,
Thanks Dominique, Stuart and Anand for sharing your experience. Yes i did ponceau S stain before probing the membrane with primary antibody. Confirmed it twice and found that it indeed doesn't impact the binding of primary antibody to the epitopes on antigen.
Before i bestow my best wishes to you all i would like to ask that which software are really good for making figures/ flow diagrams (ex cellular signaling pathways). I am currently using chem draw but if you have other suggestions please share!!
Good Luck to you all.
It will not interfere with the antibody binding. After staining you can wash with DI water for destaining . And also if you are using 5% milk +TBST then Tween of TBST ( Detergent ) will remove all the remaining staining reagent. Hope this will help .
Thanks.
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