TNF-alpha Western blot

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LJ
LJ's picture
TNF-alpha Western blot

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?

nin1318
nin1318's picture
sorry i don't know about the

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.).

RH
RH's picture
I think Santa Cruz Antibody

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.

LJ
LJ's picture
Santa Cruz sent me 2

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?

jiying huang
jiying huang's picture
Are you sure you use the

Are you sure you use the right secondary antibody?

LJ
LJ's picture
Yes, I'm using the secondary

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.

RH
RH's picture
I mean with the positive

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.

thomasdk
thomasdk's picture
LJ wrote:Yes, I'm using the

LJ wrote:

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.

Hi
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

take care

LJ
LJ's picture
My antibody is polycolonal.

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.

TRMADR
TRMADR's picture
LJ wrote:My antibody is

LJ wrote:

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.

Hi LJ,
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!

LJ
LJ's picture
I want to thank everyone for

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.

Immuno06
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Good morning from Technical

Good morning from Technical Service at Santa Cruz Biotechnology!

We want to give some feedback to the discussion in this forum.

First, we would like to advise you on our Investigator Award program. If you publish a paper citing the use of a Santa Cruz product, you may contact our Technical Service Department and receive a complimentary primary antibody!

Next, if you ever have difficulty using a Santa Cruz antibody for an application that is recommended in our product literature, please contact Technical Service. All of our products are warrantied for one year for the applications in our catalog and if a product does not perform, you can receive a credit, replacement, or refund!

Our Technical Service department can be reached at eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%73%63%62%74%40%73%63%62%74%2e%63%6f%6d%22%3e%73%63%62%74%40%73%63%62%74%2e%63%6f%6d%3c%2f%61%3e%27%29%3b')) or 1-800-457-3801 ext. 2.

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Arta
Arta's picture
I found this site looking how

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,
 
Thanks

navapaza
navapaza's picture
Hi LJ,

Hi LJ,
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!