FITC Anti-HIV1 tat antibody (ab43016)

Please find attached to this email theTAT sequence used for the epitope mapping. "TAT 1-20" corresponds to amino acids 1 to 20.

Concerning the cell localisation, we recommend to permeabilise the cells with a standard protocol using Triton or another permeabilising detergent. For more details on Flow Cytometry intracellular staining I would recommend this link : https://www.abcam.com/index.html?pageconfig=resource&rid=11448

Please find attached to this email the TAT sequence from clade B used for the epitope mapping, with reactivity scoring for different peptides. "TAT 1-20" corresponds to amino acids 1 to 20. Your nucleotide substitution is within that range and we do not know how that will affect reactivity.

Here is the sequence used for the mapping:

mepvdprlepwkhpgsqpktactncyckkccfhcqvcfitkalgisygrkkrrqrrrppqgsqthqvslskqptsqsrgdptgpke .

The first 20 aa are identical in NL4-3 Tat.

A comparison of the first 48 amino acids of Tat from several clades and NL4-3 is found in Figure 5 of the article at this link:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165250/

J Virol. 2003 August; 77(15): 8602–8606. PMID: 12857933

Thank you for getting back in contact and letting me know of your progress.

I am sorry to hear that ab63957 has as yet not produced the desired results using flow cytometry. I hope that using the new secondary antibody may provide you with better results. As mentioned in one of my previous emails, unfortunately we have not used this antibody in flow cytometry ourselves and have no reports as yet from any of our customers with how this antibody performs in this application. We cannot therefore guarantee that the antibody is suitable for this application. If you would like, I could have a look at the protocol you have been using in order to see if there are any suggestions I could make in order to improve the results observed so far. If you would like for me to do this could you please provide me with the protocol details by filling out the questionnaire I have attached to this email.

I am please to hear that you may be interested in participating in the Abcam testing discount scheme to test ab31421 and ab28837 in flow cytometry. The offer I would be able to make is our current testing discount scheme which applies to antibodies in which the application or species you intend to use have not been specified on the datasheet of the antibody. This means that we do not currently have information on if the antibody is likely to work in that particular species or application and are therefore unable to guarantee that the antibody will work. In this case, neither ab31421or ab28837 have been tested inflow cytometry. This does not mean that they would not work in this application, but we simply do not have the information in order to guarantee it.

This discount scheme works by you purchasing ab31421 and/or ab28837 (full price), and testing it in flow cytometry. Once you have tested the antibodies in this application and submitted the results through an Abreview (regardless of if the results are positive or negative) you are then entitled to a free primary antibody of your choice (or the equivalent of the original price off any product in our catalogue). The only restriction is that the antibody to be tested needs to be purchased, tested, the Abreview submitted and the free product claimed within 4 months.

More information on this offer can be found from the following link:

https://www.abcam.com/collaboratordiscount

You can participate in this scheme with either both ab31421 and ab28837 or either one individually. Please do let meknow if you would liketo participate and with whichantibody as adiscount code needs to be generated before purchase of the antibody. Could you also please let me know which cell linesyou are intending touse? From which speciesdo the originate?

I hope this information has been of help. If you require any further information please do not hesitate to contact us again.

Merci pour votre réponse.

Je me permets de vous transférer le numéro de commande de remplacement gratuit de ce produit : xx . Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

J'espère que vous auriez des bons résultats avec le nouveau tube. N'hésitez pas à nous recontacter si vous avez des autres questions.

Merci de nous avoir contactés. Nous sommes désolés d'apprendre que le produit que vous avez reçu ne fonctionne pas comme attendu.

Nous allons vous remplacer ce produit avec un nouveau tube. Par contre j'aimerai bien attirer votre attention sur le fait que nous recommandons d'éviter des cycles de congélation/décongélation (freeze/thaw cycles). Je vous conseille donc fortement, de faire des aliquots du nouveau tube, et une fois décongelée un des aliquots, le garder à 4 °C à l'abri de la lumière.

Veuillez me confirmer que vous souhaitez recevoir un nouveau tube de ab43016.

Merci de votre coopération. Je me réjouis de votre réponse.

Thank you for your inquiry and your patience.

Indeed, the questionsyou raised are very important. I have contacted the laboratory in order to find out more about this antibody.
The antibody against Tat has been generated against the full length protein. However the laboratory has mapped the epitope and there is a high reactivity on the initial amino acids. Please see the attachment for the detailed results.

The antibody specificity has been tested against
- biotinilated Red Blood Cells linked to Tat recombinant protein
- microspheres linked to Tat recombinant protein
- Tat protein internalizing human macrophages
- dendritic cells

Flow cytometry expriments can be optimised by adjusting the blocking and the amount of antibody per target mainly. To upscale the antibody signal, I can recommend to use a biotinylated secondary antibody, followed by a fluorescent conjugated strepatvidin.The secondary antibodya as well as biotin-streptavidin will enhance the signal as well. I can recommend to have a look at our online flow cytometry protocols for further tips:
https://www.abcam.com/index.html?pageconfig=resource&rid=11381
https://www.abcam.com/index.html?pageconfig=resource&rid=11443
https://www.abcam.com/index.html?pageconfig=popular_protocols

I would like to reassure you also, that we do guarantee all our products to work as stated on the datasheet (https://www.abcam.com/abguarantee).

I hope this information is helpful. Please do not hesitate to contact us again should you have any additional questions.

I'm sorry I was not available to deal with your query yesterday. I have generated the 50% discount for ab63957 as discussed which will be sent in a separate email. Let me know if you do not receive it. The testing discount code for this antibody is: 100%ABR-T7TY8 which expires on the 6th of August, 2012. In order to redeem this offer. Please order the antibody, test it in flow cytometry and tell us what you find through publishing an Abreview, putting the code given in the "Additional comments" section. Once this has been received you can place your order for the free primary antibody by placing your order in the usual way and quoting the discount code: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. The terms and conditions of this offer can be found here: www.abcam.com/collaborationdiscount. I am also able to offer a testing discount for anti-Jun B (ab31421) and anti-Jun D (ab28837) to be tested in Flow Cytometry, could you just confirm with what species you intend to use these antibodies with and I will send them to you directly.

Thank you for letting me know of your progress. I am still waiting for a response as to the exact epitope recognised by this antibody, it may be as we discussed over the phone that the antibody is not able to recognise the portion of Tat-protein which you have taken to use as your fusion. Unfortunately as the datasheet states that the antibody recognises the full Tat protein I am unable to offer a free of charge replacement of your antibody. As I understand your disappointment and the effort you have gone to in order to try and make it work, I can offer as goodwill gesture a 50% discount on the alternative antibody ab63957. However, this antibody has not been tested in flow cytometry as yet and it cannot therefore be quarenteed to work I am also able to offer a testing discount. This will entitle you to a free primary antibody of your choice if you submit an abreview with the results of your experiments with ab63957(whether positive or negative). I am sorry that I could not be of more help in this instance. I look forward to hearing how you would like to proceed.