Hello everyone, I'm currently trying to detect the expression of my target protein through Western blotting. The protein I'm studying has a size of 13 kDa and is tagged with a Flag tag. I've been using a 15% polyacrylamide gel for the separation. However, I'm encountering any signal and expression in my results. For the Western blot, I performed an overnight wet transformation. I have been using 5% skim milk as a blocking agent, and the primary antibody I'm using is anti-mouse. Thank you in advance
Do you validate your primary antibody by loading some positive control? 13 kDA is quite light, so should be transferred to membrane with no problem. Maybe slightly decrease time of gel running?
What do you expect the expression to be endogenously? How much protein are you loading? I had a 25kDa protein that I had to load a ton of protein to detect in WB (40ug/well).
The only way I was able to detect our flag tagged antibody was to also use an overexpression flag tagged plasmid to transfect cells beforehand. Then I used flag tagged beads to pull down the protein, otherwise I could not observe protein bands.
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