Hi, I am having trouble with my westerns.....my transfer is good but my protein samples don't show up AT ALL after exposure but my protein marker bands do? Any advice?
Lot of possibilities here.
If you're using fluorescent blots, the ladder will almost always show. So I'll assume transfer is perfect (no bubbles on the bands) and your sample definitely expresses the protein.
This isn't comprehensive, just a few starting points.
try to load 20 ug of proteins per well. This is the standard. If there will be no band, it means that your protein is lowly expressed. You can use a more sensitive detection substrate like Femto.
Either what Hamadi Madhi said, or your antibodies have degraded. You can try using fresh antibody dilutions.
Lot of possibilities here.
If you're using fluorescent blots, the ladder will almost always show. So I'll assume transfer is perfect (no bubbles on the bands) and your sample definitely expresses the protein.
This isn't comprehensive, just a few starting points.
Hey,
Along with all the suggestions, I would also try to check the efficacy of protein transfer to determine if proteins have migrated uniformly and evenly by coomassie staining of the gel or Ponceau staining of the membrane to check the protein transfer.
If your protein concentration is less normally, also try increase in exposure time during visualization and imaging. I got into troubles because of it so may be worth trying.
Good luck!
I would suggest: trying out few more positive controls using the same antibody. If it works, then the problem is not caused by malfunctional antibodies. Also, perhaps your protocol is suboptimal? You may wish to modify the washing step (number of washes and duration) of western blotting.
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