Scientist Solutions: Life Science Discussions
 Refer a Friend    Link To Us    Bookmark Us       

      
 » Home » Antibody Based Technologies » Monoclonal & Polyclonal Antibodies » TNF-alpha Western blot

Other Topics
11/19/2008 03:08 PM
Heat Shock proteins 90
11/17/2008 10:38 AM
beta-galactosidase ab
11/16/2008 04:17 PM
I am looking for a VSOR a ...
11/1/2008 12:40 AM
polyclonal antibody ..hel ...
7/30/2008 10:08 AM
Help with Western Blot!
7/28/2008 09:06 AM
Analysis of Monoclonal An ...
7/14/2008 04:54 AM
Search engines for findin ...
5/14/2008 11:30 AM
about Epitomics company
4/8/2008 10:45 AM
transgenicity
4/2/2008 10:03 AM
Taxonomy Viewer
3/18/2008 03:46 AM
Ryanodin receptor
2/14/2008 10:32 PM
humanized antibody
11/19/2007 04:34 PM
manoclonal antibody class ...
11/8/2007 04:47 PM
murine Activin A ELISA/W ...
8/14/2007 11:25 AM
antibodies against whole ...
8/9/2007 03:41 PM
Wondering
7/30/2007 03:14 PM
Conjugating antibodies
7/18/2007 07:30 PM
mouse IgG subclasses
7/12/2007 12:51 PM
antibody inquiring
4/8/2007 05:50 PM
Looking for Phospho-PERK ...
3/20/2007 10:27 PM
Immunoprecipitation Contr ...
2/27/2007 09:56 PM
RhoA activation of citron ...
1/11/2007 08:38 PM
Specol
12/10/2006 10:02 PM
MAB expression
9/18/2006 04:08 AM
phospho specific antibodi ...
6/14/2006 09:17 PM
Endostatin antibodies?
3/19/2006 03:50 PM
Question: What are good s ...
1/26/2006 12:05 AM
Human monoclonal antibodi ...
12/22/2005 06:21 PM
Immunocytochemistry Quant ...
12/1/2005 03:46 PM
MAbs purification by AKTA ...
Subscribet to topic
Add Reply  Add New Topic  Add New Poll
bottom of page RSS Feed 

Topic Feed

 

TNF-alpha Western blot

 [View Printable]
LJ

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 5
Joined: Apr 06, 2005







 Send a personal messsage to LJ Reply with a quote from this post Go to the top of the page

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?

.........................

 Posted Apr 06, 2005, 22:43 PM
nin1318

Frog Laureate

See
Similar
Scientists





Group: Member
Posts: 117
Joined: Apr 05, 2005







 Send a personal messsage to nin1318 Reply with a quote from this post Go to the top of the page

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.).

.........................

Posted Apr 07, 2005, 19:40 PM
RH

Tadpole

See
Similar
Scientists





Group: Member
Posts: 26
Joined: Mar 16, 2005







 Send a personal messsage to RH Reply with a quote from this post Go to the top of the page

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.

.........................

Posted Apr 08, 2005, 4:19 AM
LJ

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 5
Joined: Apr 06, 2005







 Send a personal messsage to LJ Reply with a quote from this post Go to the top of the page

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?

.........................

Posted Apr 08, 2005, 14:39 PM
jiying huang

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 5
Joined: Apr 08, 2005







 Send a personal messsage to jiying huang Reply with a quote from this post Go to the top of the page

Are you sure you use the right secondary antibody?

.........................

Posted Apr 08, 2005, 16:09 PM
LJ

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 5
Joined: Apr 06, 2005







 Send a personal messsage to LJ Reply with a quote from this post Go to the top of the page

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.

.........................

Posted Apr 08, 2005, 16:24 PM
RH

Tadpole

See
Similar
Scientists





Group: Member
Posts: 26
Joined: Mar 16, 2005







 Send a personal messsage to RH Reply with a quote from this post Go to the top of the page

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.

.........................

Posted Apr 09, 2005, 6:17 AM
thomasdk

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 1
Joined: Apr 20, 2005







 Send a personal messsage to thomasdk Reply with a quote from this post Go to the top of the page

LJ said:
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

.........................

Posted Apr 21, 2005, 0:28 AM
LJ

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 5
Joined: Apr 06, 2005







 Send a personal messsage to LJ Reply with a quote from this post Go to the top of the page

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.

.........................

Posted Apr 22, 2005, 23:00 PM
TRMADR

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 8
Joined: Oct 13, 2004







 Go to homepage of TRMADR Send a personal messsage to TRMADR Reply with a quote from this post Go to the top of the page

LJ said:
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!

.........................

Posted May 04, 2005, 12:45 PM
LJ

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 5
Joined: Apr 06, 2005







 Send a personal messsage to LJ Reply with a quote from this post Go to the top of the page

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.

.........................

Posted May 04, 2005, 10:41 AM
Immuno06

Frog Egg

See
Similar
Scientists





Group: Member
Posts: 1
Joined: Jun 16, 2006







 Send a personal messsage to Immuno06 Reply with a quote from this post Go to the top of the page

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 scbt@scbt.com or 1-800-457-3801 ext. 2.

Thanks for using our products! We appreciate your business, and we value your feedback and suggestions!

.........................

Posted Jun 16, 2006, 17:07 PM
top of page Add Reply  Add New Topic  Add New Poll

Forum Jump