I'm using HRP conjugated Antibodies and DAB based revealing buffer with 0.03%NiCl2.
Is it usual not to get the MW marker signal after adding Ponceau S? I use the manufacturer recommended volume. Once again, would a longer transfer time improve the ammount of protein transfered? I performed a 1 hour transference.
The detection limit for Ponceau S is about 250 ng/band. This amount is somewhat variable depending on the specific protein being stained. I use the manufacturer recommended volume.
What detection method is this recommended volume intended for? It may work for Coomassie staining of the gel, for example, but be insufficient for Ponceau S on a blot. You could try a titration of the markers, starting with the recommended volume and also 2x and 4x volume.
Have you consulted the instruction manual for the transfer apparatus to make sure you are doing everything according to the recommendations? For example: are you using the correct buffer (without pH adjustment); are you starting with cold transfer buffer and using the cooling block; are you pre-equilibrating the gel in transfer buffer; are you using nitrocellulose with 0.2 µm pore size (0.45 µm will also work but the smaller pore size will catch more of the proteins)?
If everything is done according to the recommendations, 1 h at 100V should be fine. If you want to go longer, you can do it overnight, at 30V, in the cold room. Going longer than 1 h at 100V risks overheating.