I am looking for TNF-alpha in rat skeletal muscle after tramatic injury via western blotting. I am not picking up TNF-alpha (17kDa) with my Santa Cruz antibody. I am picking up bands at 25kDa, 50kDa, and 75kDa. Does anyone have any suggestions?

sorry i don't know about the abundance of that protein, but is it possible that it's at a really low concentration relative to the rest of the proteins present? if that's the case, maybe try bumping up the protein concentration. or if you have alot of the samples do a concentration curve of the amount of protein and keeping the antibody concentration the same. this is usually better at finding the optimal signal since if there is too little protein there you'll never see it no matter how much antibody you add...and will be more likely to see non-specific bands (with increased ab conc.).

I think Santa Cruz Antibody not good all the time!
I did buy some Antibodies form them and I didn't get result.
but when they sent me new one " for free" I did get result.
also try to use milk with first and second antibody .
try to put positive control to check the antibody.

Santa Cruz sent me 2 additional antibodies to try. I use a 5% milk solution in PBST with the primary antibody and 3% milk solution in PBST with the secondary antibody. When you say positive control do you mean a competative binding assay? If so, I tried it and the bands remained on my samples but disappeared in my standard lanes. If you mean something else could you explain it in more detail?

Are you sure you use the right secondary antibody?

Yes, I'm using the secondary antibody santa cruz suggested. Do you think my 2nd antibody might be bad? I was using abcam primary & secondary antibodies, but they did not have the technical support so I swtich to santa cruz. I was picking up simular bands with the abcam antibodies.

I mean with the positive control to run with the sample a protein from a tissue which has TNF-a. Also, sometimes you can try to do a blotting without putting milk in first and second antibodies and put BSA instead of milk "I mean no milk". If it doesn't work the easiest thing is just to buy a new antibody from a different company.

My antibody is polycolonal. I have several time points, 1.5, 3, 6, 9, & 12 hrs post injury. From 3 papers, control values of TNF-alpha have been reported for muscle.

I want to thank everyone for their suggestions. I just got it to work yesterday. I am loading more total protein; using a PVDF membrane; using a different primary antibody from Santa Cruz (N-19 rather than L-19); have decreased my secondary antibody concentration; and have increased my milk concentration to 5% in the secondary antibody solution.

TRMADR - I had tried using a cell lysate as a positive control and wasn't getting a signal with the L-19 or the N-19. But I have confirmed the bands in my tissue samples are TNF-a with acompetative blocking assay. Thanks.

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