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The reply below was sent to me by one of supervisors, Dr.Davies who has been involved with my problems with the antibodies purchased, I assume someone else picked up my faxes then and sent response to him instead of me seeing his name was on the purchase order.
Your advice seems somewhat different from the ab tech support below esp. re Glycine but I have followed your instructions instead on the 2 antibodies (titration, wash, incubation) and still unable to get any immuneflourescence and I am no longer willing nor have the time and resources to validate ab49873 and ab93434 the antibodies which are supposedly validated by the company and under ABGUARANTEE.
As both these antibodies have not responded please replace them for me instead of refund:
As we have discussed before please send me the L glutaminase ab 914073 as your gesture of good will,
I will also need ab 60709(K glutaminase as per your new updated sheets) and ab 16802 (glutamine synthetase),
Could you please replace the 2 antibodies under abguarantee which have not worked with the 2 that have worked so we can get to a closure of this long winded ordeal for me?
Asked on Mar 22 2012
Thank you for getting back to me.
I am sorry that the reply to your fax was sent to your supervisor by one of my colleagues, Keith. This was due to the way our assigning system works. The protocol tips that I gave and those of Keith are not that dissimilar. We both recommended optimising the dilution of the antibody. Keith also recommended using Glycine to reduce the possible background created from uncapped aldehyde groups. I did not mention this as you were having problems seeing any staining, the background was therefore not my top priority to tackle. What may be significantly contributing to your lack of signal may be the way that the tissue is fixed. Over fixation can lead tothe epitopeto be probed to bemasked.Even with permiabilisation, thismay not be unmasked. Someform of antigen retrieval may therefore be necessary.In my previous email I tried to find out a little more about this in order to be able to provide advice on how this could be improved:
1. Exactly how was the tissue fixed? For how long was the tissue treated with PFA? What concentration of PFA was used? At what temperature?
2. Has the goat anti-rabbit secondary antibody used for both experiments been used successfully before?
3. With the tissue sections that you have, have they been successfully stained to detect a different target with a different antibody? Could you send me some images of this staining?
If you could also let me know under exactly what conditions the new experiment was carriedout, that would be helpful. Which ofmy suggestionsdid you try?
As I mentioned in my previous email, ab93434 has to our knowledge not been used in staining of frozen sections and it may be that the antibody is not suitable for this application. As this application is not listed on the datasheet the antibody is therefore not covered by the Abpromise for this application.
As you are having problems with both of these very different antibodies and I am as yet not sure what is contributing to this problem, it seems that it may either be the tissue being used or the protocol which may be contributing to the lack of signal. As I want you to get the best staining that you can, I would therefore like to ascertain if it is a problem of the antibodies first. Have you performed a western blotting experiment with these antibodies? If not, I would like to provide you with a positive control for this application to test the activity of the antibodies.
If you would like for me to send this positive control for western blotting please do let me know. Otherwise, all I can offerat this time is a refund, in addition to the ab91073 which I offered as a good-will gesture.
I hope this has been of help. I look forward to receivingyour reply.
Answered on Mar 22 2012