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.
Have you checked whether its monoclonal or polyclonal antibodies you have ? ,- cause if its monoclonal, the epitopes might be hidden until you microwave the tisssue samples and unfold the epitope. Also, how long time do you alow between crushing and taking out the samples you need ?+
p.s. DAKO have good aBs
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.
It sounds as though you have done quite a bit of homework and trouble shooting. I would suggest a "second opinion". that is to say, a different primary antibody. The antibody search tool on biocompare has some functionality, but a quick google search for this specificity may also work. I believe there is a commercially available clone MPX something or other that you might consider. Without saying too much, my impression is that some companies in the antibody industry seem to focus on "speed to market" while others focus on "quality of product". Being that TNF-a is a fairly well characterized molecule, your best bet might be to try another source. Have you tried any "positive control lysate", which is run side by side with your experimental lysate? There are probably several readily available tissue or cell lysates that are known to contain detectable levels of TNF-a. Just a couple thoughts. Good luck with that one!
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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I found this site looking how to isolate TNF a in mice heart after ischemia. I saw that it did work for you to detect TNF in musle, I would appreciate if I could get your protocol, in particular how did you homogenize and spin the tissue,so the complete protocol would be very helpful,
perhaps you can still remember your problems with TNFalpha Westerns... well, I have got the same. Would be very nice of you if you could give me further hints. Thank you so much!