I'm trying to run an SDS-PAGE with a Western Blot. The SDS-PAGE is working fine so far, but I'm not sure if the Blotting really works. I'm using Nitrocellulosis and run the gel at 100 V for 1 h.
To prevent overheating of my Transfer buffer, I'm using cooling pads in and around the chamber. After 1 h I see the bromophenol blue of my prestained marker on the NC Membrane. When I now stain the Membrane with Ponceau S I cannot see any bands on the NC. (That could possibly be caused by too low concentration? So low that it is under the detection limit of the Ponceau method?).
In the following I'm trying to make the bands of interest visible. For that I'm using materials of an ELISA-kit. In detail, I have an antibody on the NC membrane, I'm giving specific antigen (from the ELISA kit) on the NC, then I'm adding specific biotinyled detection antibody and strep-HRP (also from the ELISA kit). When I'm now adding TMB (HRP-Substrat) the solution turns blue step by step but I cannot see any changes of the NC membrane itself. The positive control I'm using is primary antibody (5myg/ml), Antigen (1 ng/ml), secondary AB (1 myg/ml).
Is it possible that maybe only the blotting of bromophenol blue of the marker works but because of any reason no Protein is blotted? Is the voltage or time of bllotting too high/low?
Thank you very much