Anti-Bax antibody [6A7] (ab5714)

phone call: very low signal (maybe half intensity) in WB with this new batch used also at 1/200

ANSWER:

Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que le nouveau lot de l'ab5714 que vous avez reçu ne fonctionne pas comme attendu. Je me permets de vous transférer le numéro de commande de remplacement gratuit de ce produit avec un nouveau lot : xx. Ce produit devrait arriver demain chez vous. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

Je serais ravi d'avoir de vos nouvelles pour savoir si le nouveau lot fonctionne bien. Aussi, comme discuté au téléphone, je vous conseille de garder le tube à 4°C. Si vous voulez tester la congélation de cet anticorps, et après congélation il ne fonctionne plus, je peux aussi - en échange des résultats, le remplacer de nouveau.

N'hésitez pas à nous contacter si vous avez des questions.

Je vous souhaite une bonne journée et des bonnes pâques!

Recently, we have bought from you the mouse monoclonal [6A7] to bax (ab5714).
As secondary antibody, we would like to use the Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Chromeo™ 546) (ab60316).
We are working with PC12 cells (rat pheochromocytoma).

Are these antibodies compatible?

ANSWER:

Thank you for contacting us.

Yes, those two antibodies, ab5714 and ab60316, are a compatible pair, and ab5714 will recognize rat Bax.

Please note that ab5714 should not be stored frozen, but only at 4oC.

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

Hi xxxxx,

From previous western blots, it does not appear that the protein I am working with (Capsid) causes any changes in Bax expression. It is pulled down by Bax in immunoprecipitations. In my protocol I fix cells with 4% paraformaldehyde for 30 minutes at room temperature before quenching in 50mM NH4Cl for 5 minutes. I have included two pictures. One gives example pictures from the working stock and the most current stock I received. The secondaries are switched between these two experiments but I have seen similar results for the current stock when they were the same. The exposure time for the 6A7 in the current stock is also much higher (˜5 seconds). The Capsid protein is the protein I am studying that activates Bax. The other picture I have included is from a paper by another graduate student in our lab who was studying the same thing.

ANSWER:

Thank you for that information.


I'm sorry but I've got a few more questions for you just to clarifya few more things:

Have you performed a western blot/IP with thisnew lot?Have you tried the staining experiment with just one of the three vials sent to you or have you tried all three with the same results? The"working stock" of the image, was that the original lot you had before freezing it?


I remember you have also ordered anti-Bax antibody ab69643 previously, how have you found this to perform?


Many thanks foryour continued cooperation.

Hi,

Sorry for the late reply. The antibody did arrive, but I wanted to try a few experiments with it before responding. It seems I am still having the same problem with the antibody, where it does not seem to recognize activated Bax in cells transfected with my protein. I have noticed a speckled pattern in some cells that are dying and detaching from the coverslip, but the brightness is not much higher than background in most cells.

Thank you

ANSWER:

Thank you for letting me know of your progress. I am sorry to hear you are still having problems with this antibody.


Are you seeingany specificstaining ofBax in your cells (untransfected ortransfected)?Have you triedperforming western blotting or immunoprecipitation to see if your transfected construct increases the Bax expression?


Am I correct in thinking you have previously seen good staining with the antibody with one of your previous lots (before freezing)? Would you be able to share the images obtained with the different lots with me please?


You have described your protocol very clearly to me before, could I just check how long you are fixing the cells with the paraformaldehyde and at what temperature is this step being performed?


I look forward to receiving your reply.

Hi , Here are the answers to the questions you provided in your last e-mail:

1. Usually I use the antibody at a dilution of 1:50 in PBS + 1mM MgCl2 + 0.5mM CaCl2 + 1% BSA. With the frozen aliquots in question I tried smaller dilutions up to and including undiluted antibody and had no luck.

2. I incubate the antibody overnight at 4°C. The cells I use are A549 cells.

3. Prior to incubation with the antibody, the cells are washed twice with PBSCM, fixed in 4% paraformaldehyde and then quenched for 5 minutes with 50 mM ammonium chloride in PBS. The cells are then permeabilized for 4 minutes with 0.2% Triton in PBS, washed twice in PBSCM and then blocked for 15 minutes in 1% BSA in PBSCM. After this the coverslips are placed in the staining solution mentioned above.

4. I haven't been able to dig up the lot number, as the original tubes have been discarded. The purchase order number is xxxxxx and the order reference number is xxxxx. It was ordered back in August.

I have used a more recent stock we purchased and found that after thawing and refreezing it also does not seem to work for this application (although I was unable to use it directly after the initial thawing). I find that this antibody does still work well for Immunoprecipitation though.

ANSWER:

Thank you for providing those extra details. This information to investigate this case further and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and am sorry that the product has not been performing as expected.

I apologise for the inconvenience this has caused. I would be happy to offer a free of charge replacement for the two vials of the antibody you have purchased if you would like? In order to process this I would need the details of the more recent order (the one after 916535), order number or order date and delivery address.

If you would like a replacement  I would suggest storing the antibody at 4°C. But if you have any further problems please do let me know.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.