Anti-EEA1 antibody - Early Endosome Marker (ab2900)

I appreciate your help in this matter. Since I have found working conditions for this antibody (ab2900), I don’t see sense in getting any of the other anti-EEA1 antibodies since I will have to start looking for the optimal conditions again. You say that you guys have a new lot of ab2900 but has not yet tested it. Do you know when would that be? If you expect it to be tested in the next several weeks, I can wait to see if it works better than the previous lot in terms of optimal dilutions and specificity. If is not going to be tested soon, or perhaps not at all (since is coming from the same rabbit, for example), we would then chose to get another vial of the same lot.

Again, I greatly appreciate all your help during these process. As soon as I hear back, I will get you the original order number and other information you may require.

Thanks and have a nice day!

ANSWER:

Thank you for your reply and for your patience.

I apologize for any confusion, but the new lot of ab2900 has actually been tested in ICC/IF. We do test all new lots of each antibody in various applications.We just haven't tested this lot side-by-side with the previous lot, so we are not sure if it will have a higher binding affinity. At 5 ug/mL in HeLa cells fixed with methanol, the staining seems to be quite strong (see attached image).

If you approve of this lot, I'll be happy to send a free of charge vial. Please forward me your original order number if you would be interested in receiving this lot.

I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that we can do for you.

Thanks a lot for the information. Prior to see your email, I was in the progress of testing 1:200 and 1:100 dilutions, although not with the same fixation/permeabilization conditions that you mentioned. I did get some staining at these Ab dilutions (before there was no staining at all), but it wasn’t punctate, more like big patches or whole cells were stained. I do realize that because these are polyclonal, there is going to be a lot of variation from batch to batch. I’m going to try exactly these conditions that you mention, although I don’t see why similar fixatives and detergent wouldn’t work (I’m an optimist!). I appreciate your help and I will keep you updated on my results in case I still have problems with this lot, thanks.

ANSWER:

Thank you for your reply and for the additional information.

Thank you also for keeping me updated about the results using the same fixative and detergent, and I look forward to hearing from you about this. We are happy to send a different antibody or another vial of ab2900 if the staining does notseem to becorrect.

Please let me know if you have any questionsand let me know if you need anything else. Have a great day!

A few weeks back we bought from you an anti-EEA1 antibody (ab2900) which I need for immunofluorescence in THP-1 cells. I have tried several times now under different conditions and none have worked. I’m wondering if you know problems with this specific lot #1195246. I have tried the recommended concentration (roughly 1:1000 dilution) and up to 1:250, incubated 1hr up to overnight, used different detergents for permeabilization, and it never works. I would appreciate if you guys have a standard protocol specific for this antibody to let me know, or at least any comments that could help.

My project will require staining of EEA1 pretty regularly, and my first choice was Abcam which I have not had any problems in the past, but this time it looks like the antibody is not working, thanks!

ANSWER:

Thank you for your email and for your patience while I've been in touch with the lab to request the protocol used to test this antibody.

According to our in house data,this antibody was tested in HepG2 cells fixed in 4% paraformaldehyde and permeabilised in 0.1%Tween/PBS. The optimal concentration for the lot that you have triedwas 5ug/ml, which corresponds to 1:180 dilution. We’ve not tested this in THP-1 cells butwe would expect it towork.

We would recommend trying the 1:180 dilution and using 4% PFA and PBST fixation and permeabilization steps if you haven't tried those yet. If the results do not improve in these conditions, I would be happy to send a free of charge vial from a new lot, or a different antibody of your choosing.

Please keep me updated about any new results, and let me know if there is anything else that we can do for you.

Dear Sir/ Madam
I want to use ab2900 as the primary antibody to mark early endosome. Could
you please recommend me a secondary antibody which is suitable for the
primary antibody. I need it to be green fluorescence labeled. Tanks a lot.

ANSWER:

Thank you for contacting us.

ab290 is a rabbit IgG antibody. The followings are some compatible secondary antibodies whichhave been tested in ICC/IF and areDyLight 488 or FITC (green fluorescence) conjugated:

ab96883 (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (DyLight® 488))

ab96891 (Donkey anti-Rabbit IgG H&L (DyLight® 488) secondary antibody)

ab96923 (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (FITC))

ab97063 (Donkey polyclonal Secondary Antibody to Rabbit IgG - H&L (FITC))

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

We would be interested in getting free antibody samples to test two of
your antibodies on dog samples, namely Ab 61778 and Ab 2900. Would it
be possible if we give you back data? I thank you in advance

ANSWER:

Merci pour votre requête.

A notre connaissance, la réactivité contre le chien n'a pas encore été testée avec l'anticorps ab61778. Par contre l'anticorps ab2900 a lui été testé chez le chien et est donc garanti pour fonctionner chez cette espèce. Toutes les informations sur les cross-réactivités d'espèces sont spécifiées dans notre fiche technique, et sont mises à jour régulièrement.

Malheureusement, comme nous avons plus de 65.000 références anticorps à notre catalogue, vous comprendrez que pour des raisons pratiques et logistiques nous ne pouvons pas offrir de petits volumes pour des essais préliminaires.

Si vous décidez de tester le ab61778 chez le chien, nous pouvons vous proposer une offre de test en échange de vos résultats sous forme d'Abreview.
1) Nous informer que vous souhaitez tester un de nos anticorps et nous vous attribuerons un code promotionnel
2) Acheter l'anticorps à tester
3) Testez l'anticorps
4) Que le résultat soit positif ou non,envoyer vos résultats sous formed'Abreview (http://www.abcam.com/abreviews) et le code sera activé
5) Le code vous permet de commander gratuitement n'importe quel anticorps primaire du catalogue Abcam.

J'espère que ces informations vous aideront. N'hésitez pasà me contacter si vous avez besoin de plus de renseignements.