Anti-COX2 / Cyclooxygenase 2 antibody [5E10/D10] (ab701)

I bought 2 Antibodies from you recently. I made a mistake and bought the COX-2 monclonal, ab-701 (cross-reactive to human and mouse) As 2:nd Ab I choose ab6728. The problem is that I am using rats in my experiments and the ab701 seems not to crossreact with rat according to the product sheet, is that still correct? On my western blot I recieve 2 distinct bands. Is there anyway that I can confirm that this also can be used for rat? To use another primary antibody and compare the resulting bands or? Is there any COX-2 antibody for suitable for western blot that can be used together with my ab6728??

ANSWER:

Thank you for your enquiry. We are very sorry for the trouble and for not choosing the right antibody.

Please do not despair! The antibody may work in your system; it has just not been characterized for cross-reaction with rat. But it does not necessarily mean that it does not recognize rat COX2.

We would therefore suggest a couple of things:

1. Try to use a well-established positive control (from human or mouse, these two species have been tested) and run your samples along with it to be able to identify the bands.

2. As you probably know, COX2 (Cyclooxygenase 2) is an inducible enzyme. COX2 expression markedly increased in 85-90% of human colorectal adenocarcinoma.

A single band at around 72kDa is expected, however we have received a report from a customer working with this antibody that they obtain two strong bands in WB - one between 40 - 50kDa and other between 62 - 83kDa.

Are the two bands you have around these sizes?

Please do let us know how you are getting on with this antibody. Your results are interesting and we would be very grateful for you if you can submit a review about this antibody once you are confident with the results. We can update our on-line datasheet with your findings and in return we can we will send you an Amazon voucher.

Good luck with your experiments!

Thanks for your email and the offer to replace the antibody. Although I would like to try the antibody to Cox 2 again. I am afraid that the same problem would occur again. If you have antibody to Alpha 3/ beta 1 integrin that reacts with human cell lines in both ELISA and Western Blot, you can provide me that antibody, if not, please give us the refund. If you like for us to send you back your anti- Cox, I will be happy to do so.

ANSWER:

We currently don't have an integrin beta 1 antibody that has been tested for application in both ELISA and Western blotting. I have contacted our accounting department and instructed them to issue you a refund for ab701. If I can be of further assistance, please do let me know.

Thank you for your email. The antibody that we purchased from abcam is: Mouse monoclonal antibody (5 E10/D10) to Cox2 (Cyclooxygenase2) 50 ug ( 1 mg/ ml), abcam- 701-50, Lot # 48755. The followings are my response to each of your question

Q #1. We get no signal . Q#2. Indirect ELISA

Q#3. Human Cancer Cell Lines. Tumor cell lines were propagated into 96- well plates, fixed with glutaraldehyde (0.1%) for 5 minutes, washed 3X with PBS and blocked with glycine (5 minutes) , washed with PBS and treated with media for at least 1 hour before tested with ELISA.

Q#4. See above explanation.

Q#5. Mouse monoclonal antibodyto Cox 2 ( your antibody, 5 E10/D10) Different dilutions were used. First used 0.5-0.1 ug/ml dilution as indicated in your data sheet. Since this dilution did not give any positive reaction, lower dilutions were tested and the results were negative again. After incubation we wash each well 3X with PBS.

Q#6. We have tried Goat anti mouse beta galactosidase-conjugated as well as Goat anti mouse alkaline phosphatase-conjugated secondary antibody. Neither one has given any positive results. We routinely use 1:500 to 1:1000 dilution of our secondary antibodies. They have been standardized in our laboratory. Incubation time for us is two hours for both primary and secondary antibody and wash each well after incubation 3X with PBS. Our secondary antibodies has been tested many times and work very well. I don't think there is any problem associated with our secondary antibodies.

Q# 7. We use p-Nitrophenyl-beta- D-galactopyranoside as substrate for beta galactosidase conjugated secondary antibody and and p-nitophenyl phosphate for the alkaline phaspatase conjugated antibody.

Q#8. Yes, we always include positive and negative control in our tests. I used mouse monoclonal antibody to human alpha 3 beta 1integrin as a positive control and a mouse anti bacterial monoclonal antibody as a negative control.

Q# 9. We have tried 3X with each different secondary antibody using different dilution of the anti Cox antibody and we have always had negative results. We have also used different cell lines with similar results.

I should mention that we conduct ELISA, Western btot and other immunological assays in my lab on daily bases. All of these tests have been standardized in my lab. I believe that either wrong antibody was sent to us or your antibody is not working. In either case, I am disappointed. I hope you can respond to this email and let us know if you have any suggestions.

ANSWER:

Thank you very much for the details that you have provided and for your patience. I have contacted the originator of this antibody and am waiting to hear if there have been any other complaints regarding the lot that you have received. As we have had no other complaints concerning ab701, my feeling is that there very well may be a problem with the lot/vial that you received. We do have a newer batch in stock and I can send it to you as a free of charge replacement. Alternatively, I can offer you a refund or a different antibody to try. Please let me know which you would prefer, and once again I thank you for your patience.

Dear sir/madam I am trying to find a Mouse anti Rat Cox-2 primary antibody. I note you have a mouse primary antibody (ab701). The product data sheet claims this has not been tested on Rat. Is this still the case? If so, do abcam have any mouse primary Cox-2 antibodies that are known to react with rat Cox 2?

ANSWER:

There has been no further information for the antibody's cross reactivity with rat. We do not have another mouse COX 2 in our range. If you cannot find what you are looking for within the Abcam catalog, I suggest that you click on the link to The world's Antibody Gateway. The world's Antibody Gateway is a free search engine service provided by Abcam to help you to quickly find the antibodies that you are looking for. It is a free-text search engine developed by the Abcam team so that it searches the catalogs of all online antibody companies (currently 249).