I've been having issues visualizing blots when probing for the Myc tag in HEK cells. I'll list my basic step outline that I follow, maybe that will be informative in picking out something I'm doing wrong:
Gel electrophoresis, transfer -> Either block membrane in 5% milk in 1x TBST at 4 degrees overnight or block at room temperature for an hour -> Incubate overnight with primary antibody (Mouse Myc antibody) at 4 degrees; 1:1000 in 5% milk in 1x TBST -> Wash membrane 3x for 15 minutes in 1x TBST -> Incubate in secondary antibody (donkey α mouse) at 4 degrees; 1:5000 in 5% milk in 1x TBST -> Wash membrane 3x for 15 minutes in 1x TBST -> Drip on HRP, wait 3 or so minutes, remove excess HRP and put an extra film on top of the membrane so it doesn't dry out -> Visualize in chemidoc -> See nothing.
Check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466278/
Also check https://www.labome.com/method/Protein-Peptide-Tags.html
Question: did you include a positive control in your blots? Do you get a signal with them?
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