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Thank you for your reply. Here you find the answers to your questions:
1. The order number is ###
2. In the attached file you can see some images of the blots we have. For each image you can find blot conditions.
3. We generally use milk solution to block the aspecific sites and we’ve never had problems. In our case, I guess the problem is not a high aspecific signal (which we could see only in our samples with a big increase of the image contrast) but the absence of bands, in our samples and also in the positive controls. Anyway, in your site I found that another research group performed very similar experiments using milk as blocking agent (reference: https://www.abcam.com/Cyp3a1-antibody-P6-ab22724.html&tab=abreviews&intabreviewid=7693).
4. The microsomal fractions are prepared according to a method of differential centrifugation as described in:
- Floreani M, Gabbia D, Barbierato M, DE Martin S, Palatini P., Drug Metab Pharmacokinet. 2012 (in press). Differential inducing effect of benzo[a]pyrene on gene expression and enzyme activity of cytochromes P450 1A1 and 1A2 in Sprague-Dawley and Wistar rats.
Recombinant rat CYP3A1 microsomes are purchased from BDGentest and are used as positive control, since they are microsomes which express almost exclusively our protein of interest. Using this type of positive control usually permit to avoid the risk of unspecific signal, which is common with whole cell lysates.
Both samples of microsomes were prepared with Laemmli loading buffer (4% SDS, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris HCl) even with or without 10% b-mercaptoethanol, and then boiled for 5 minutes.
5. We used two different secondary antibodies that have been successfully tested with other primary antibodies (anti b-actin, anti cyp1A1/1A2 and anti AhR antibodies-reference: Floreani M, Gabbia D, Barbierato M, De Martin S, Palatini P., Drug Metab Pharmacokinet. 2012, Differential inducing effect of benzo[a]pyrene on gene expression and enzyme activity of cytochromes P450 1A1 and 1A2 in Sprague-Dawley and Wistar rats.)
6. The antibody was used the first time the day of arrival and then stored as described in the datasheet.
I hope my answers are clear and exhaustive. Please let me know if you have further suggestions. Otherwise, since I have seen two polyclonal antibodies anti cyp3A1 (ab22733, AntiCytochrome P450 3A1 antibody Rabbit polyclonal; and ab22732, Anti-Cytochrome P450 3A1 antibody Sheep polyclonal), I wonder if you can provide us a credit note to try one of them. They look similar to the CYP3A2 one, that works very well in our samples and conditions.
Looking forward to hearing from you,
Asked on Oct 17 2012
Thank you for your message and for providing this further information.
I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or credit note in compensation.
I look forward to hearing from you with details of how you would like to proceed.
Answered on Oct 17 2012