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Dear Abcam tech support,
Please advise on the following enquiry posted by our customer:
I'm preparing some western blot experiments using the human serum or plasma hypersensitive to the allergen of interest to a recombinant
allergen I've made. The steps for the western blot that I have
planned are as follows:
(1) Run SDS-PAGE of the recombinant allergen protein
(2) Transfer the allergen protein from the SDS-PAGE gel to a nitrocellulose membrane
(3) Incubate membrane with human serum/plasma known to be hypersensitive to the allergen (human IgE binds to allergen on the
(4) Incubate membrane with mouse/rat/rabbit anti-human IgE antibody which will bind to the human IgE
(5) Incubate membrane with anti-mouse/rate/rabbit anti-IgG conjugated with HRP (secondary antibody)
(6) Visualise result with ECL.
My questions are as follows:
(a) would the mouse monoclonal to human IgE (ab7382) be appropriate?
If so, could you let me know the price for this antibody?
(b) do you have a secondary antibody conjucated to HRP that I could use for step 5? If so, could you let me know the price as well?
The reason why I'm doing steps (4) and (5) instead of just step (4) with an anti-IgE conjugated to HRP is that I've been told that as IgE is usually in very low abundance in human serum/plasma, thus I would probably be unable to detect the IgE directly without doing step (5).
But I'm not sure if there are sensitive antibodies that could do away with step (5). I have read some papers where people have skipped step
(5) for Western Blot. Do you have a product that is sensitive enough so that I can skip step 5 altogether? If so, could you let me have the quotation as well?
In the meantime, have a nice day!
Asked on Aug 30 2012
Thank you for contacting us and sorry for the delay in getting back to you.
In answer to your questions:
1. Yes, I believe the antibody ab7382 should be suitable for the experiment you have described, however, as we have not performed a Western blot in this way (directly binding the human IgE to the membrane), we would not know exactly how the antibody would perform. It may be that you would need to perform some optimisation of the protocol in order to obtain the best results.
2. The goat anti-mouse IgG1 (https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Mouse-IgG1-heavy-chain-HRP-pre-adsorbed-ab98693.html) should be suitable for the experiment you are planning. Sapphire will have to inform you of what the price of this will be.
In answer to your question of if the directly conjugated, ab7294 could be used instead of using the primary ab7382 followed by the secondary antibody, I would suggest this would be possible but as you have said the signal would be weaker, so if the target is not abundant then using a secondary antibody would be beneficial. Additionally, as we have only used ab7294 in ELISAs as yet, we would not be able to know how it would perform in a Western blot. I would therefore suggest using ab7382 with a secondary antibody may be more appropriate.
I hope this information has been of some help. If you have any further questions, please do not hesitate to ask.
Answered on Aug 30 2012