Anti-Tenascin C antibody [BC-24] (ab6393)

ab3970 (Tenascin antibody [T2H5]) ab6393 (Tenascin antibody [BC-24]) ab7839 (Tenascin antibody [B324.1.])

Do these products recognize tenascin C?

ANSWER:

Thank you for your patience.

I have contacted the originators of ab3970, ab6393, and ab7839 and have received the following replies:

ab3970 - This antibody reacts with tenascin; it was raised against mammary tumor of human origin. Tenascin-C could be the alternate name of tenascin: Tenascin-C (Tn) is the best characterized member of the tenascin family of cell-adhesion proteins - Alternate names of tenascin C: tenascin; cytotactin; hexabrachion.

ab6393 - The epitope recognized by this antibody is common to all Tenascin isoforms. Thus, the antibody will recognize Ten-C along with the other isoforms of tenascin.

ab7839 - No reply. I have recontacted them.

I hope this information helps. If I receive additional information on ab7839, I will e-mail it to you.

Please do not hesitate to contact us again if you have additional questions.

The customer is looking for antibodies to use in IHC-P (IF) on chicken embryos against: -tanascin -myosin heavy chain

ANSWER:

Thank you for your recent phone call and your interest in our tenascin and myosin heavy chain antibodies.

As we discussed we do not currently have antibodies which have been tested in chicken tissue and the homology searches revealed that for tenascin there is 64% identity between the human precursor form (immunogen of our tenascin antibodies) and the chicken precursor form. This is not extremely high and therefore our antibodies do not have high chances of cross reacting with chicken tissue. You may find better antibodies for chicken tissue on biocompare.com or at the bottom of the search for tenascin on our website a link to the World's antibody gateway which looks at 149 other online antibody companies.

I am trying to find out more information about the immunogen sequence for the myosin heavy chain antibodies to work out the homology between the immunogen and the chicken protein, my apologies for the delay.

Thanks a lot for your answer. Concerning your questions: 1. The transfer was good. 2. Other people in our lab work with the same secondary antibody and detection system and they work well.

In the meantime I've tried once again the western blot with a purified human Tenascin C as positive control: it worked perfectly! But my tumor samples are still negative, whereas they are strongly positive in immunohistochemistry...

Thanks a lot for your help!

ANSWER:

Thank you for your reply and providing more details.

As I understand, you have used purified human Tenascin C (positive control) and the antibody recognized the target; however it failed to work in the samples. Do the pancreatic tumour cells express Tenascin? If yes, at what levels? Have you tried to load a bit more protein than 30 ug/lane to see if the signal is getting stronger?

ANTIBODY CODE ab6393 BATCH NUMBER 12034

DESCRIPTION OF THE PROBLEM No signal

SAMPLE human pancreatic cancer cell lines

PRIMARY ANTIBODY ab6393 1:1000 to 1:4000 O/N 4?C in TBS-T with 2% non-fat dry milk

washing: 3x 20 min in TBS-T

SECONDARY ANTIBODY Amersham goat anti-mouse 1:3000 1h at RT in TBS-T with 2% non-fat dry milk Washing: 3x 20 min in TBS-T

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED positive control: human pancreatic cancer tissues

ANTIBODY STORAGE CONDITIONS -20?C

SAMPLE PREPARATION 10% SDS Glycerin Tris HCL 2-Mercaptoethanol Complete Mini protease inhibitor cocktail tablets (Roche)

sample boiled for 5'

AMOUNT OF PROTEIN LOADED 30 ?g

ELECTROPHORESIS/GEL CONDITIONS 7.5%, not reducing

TRANSFER AND BLOCKING CONDITIONS 90 min transfer, Tris-Glycin-SDS

Blocking buffer: 5% non-fat dry milk TBS with 0.5% Tween (TBS-T)

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? dilution of the primary antibody

ANSWER:

Thank you for getting back to us and providing more information about your assay.

We have read through your protocol very carefully and would like to ask two more questions which could help us to identify the source of the problem.

1. Has the protein transfer been sufficient? Could you see the molecular weight markers clearly on the nitrocellulose membrane?

2. Have you tested the detection system? It is always important to test the secondary antibody with another primary antibody to make sure that the detection system works properly.

Thank you for your co-operation in advance. We are looking forward to hearing from you soon.

Good evening,

I would like to know if someone used this antibody with rat tissue. So, could you let me know if you have any information about this. Thanks a lot

ANSWER:

This antibody has only been tested against the human antigen. Recognition of rat tenascin is unknown.