When we add Ponceau Red to the fresh transfer blot we could see the protein transferred to the membrane, but once that membrane was stripped by a harsh method, we could not see the protein bands in the membrane.
Note we followed abcam method of harsh stripping for this.
Most of the proteins are still blocked by the blocking solution. If not, you have stripped too much.
Hi, I've been using similar harsh stripping protocols before and they normally stripped your proteins, so probably that is the reason you do not see your proteins. Normally, even the membranes still blocked, you can see bands (they normally look quite dirty because the blocking, but you still able to see the most abundant ones). I normally do not like to use harsh stripping protocols for membranes I'm thinking to reuse. First, because the high risk remove all proteins and, second, because if you do not remove all of them you will remove a quite unknown amount of them… and I personally do not like that. If you really have to strip your membranes there are pH based stripping protocols (that do not need high temperatures and ß-mercapto) that can remove the primary and secondary antibodies quite efficiently. Also, if you have to detect another protein with a quite different molecular weight probably you do not need to strip the membrane. Hope that helps.
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