Anti-Cyp3a1 antibody [P6] (ab22724)

Reviews (2)Q&A (4)References (6)

Overview

  • Product name

    Anti-Cyp3a1 antibody [P6]
    See all Cyp3a1 primary antibodies

  • Description

    Mouse monoclonal [P6] to Cyp3a1

  • Host species

    Mouse

  • Tested applications

    Suitable for: Flow Cyt, ELISA, WBmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human, Pig

  • Immunogen

    Full length protein corresponding to Rat Cyp3a1.

  • General notes

     This product was previously labelled as Cytochrome P450 3A1

     

Properties

Applications

Our Abpromise guarantee covers the use of ab22724 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt Use 1-2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 
ELISA Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 57 kDa.

Target

  • Relevance

    In animals, P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs. The four major families involved in drug metabolism are CYP 1,2,3, and 4. Cytochrome P450 3A4 [human-3A1 Rat] is abundantly expressed in liver and small intestine and is inducible by barbiturates, glucocorticoids and rifampicin.

  • Cellular localization

    Endoplasmic reticulum, membrane bound.

  • Database links

    • Alternative names

      • CYP3A1 antibody
      • cytochrome P450, family 3, subfamily a, polypeptide 1 antibody

    Images

    • Flow Cytometry - Anti-Cyp3a1 antibody [P6] (ab22724)
      Flow Cytometry - Anti-Cyp3a1 antibody [P6] (ab22724)

      Overlay histogram showing HepG2 cells stained with ab22724 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab22724, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

    References

    This product has been referenced in:

    • Gabbia D  et al. Pregnane X receptor and constitutive androstane receptor modulate differently CYP3A-mediated metabolism in early- and late-stage cholestasis. World J Gastroenterol 23:7519-7530 (2017). WB ; Rat . Read more (PubMed: 29204052) »
    • Qin Y  et al. Comparison of Pharmacokinetics and Tissue Distribution Kinetics of Roxithromycin and Expression of CYP 3A1 between Pregnant Mice and Foetuses. Basic Clin Pharmacol Toxicol 120:146-151 (2017). WB ; Mouse . Read more (PubMed: 27611991) »
    See all 6 Publications for this product

    Publishing research using ab22724? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-6 of 6 Abreviews or Q&A

    Question

    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/index.html?datasheet=22724&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,

    Best regards

    Read More
    Answer

    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.

    Read More

    Question

    Inquiry: We performed several western blots with this antibody following these conditions: -
    Rat liver microsomes: 50-20 or 10 ug - Recombinant rat CYP3A1 microsomes: 10-2.5-0.25-0.125 pmoles.
    -Blocking condition: 10% milk in TBST 1 h, RT
    - Luminata (Millipore) or ECL Plus (Amersham) as HRP conjugated detection systems, using a Versadoc (Biorad) detector
    - SDS-PAGE 8% PAA, blot on a nitrocellulose membrane. Protein samples were prepared in denaturing and reducing condition (beta-mercaptoethanol) and, subsequently, in denaturing and non-reducing conditions (as you described for this antibody).
    - Primary antibody: dilutions used: 1:2000, 1:1000, 1:250, 1:100 in 5% milk in TBST. Incubation was performed ON at 4 deg.
    -Secondary antibody: dilution used 1:2000, 1:5000 in 5% milk in TBST. Two different secondary antibodies were tested without any difference in results. Incubation was performed for 1,5 h, RT.
    -Wash steps: 3 times for 10 minutes after blocking, primary antibody and secondary antibody incubation.
    We performed the same experiments with Anti-CYP3A2 (ab22731), obtaining perfect results with primary antibody dilution of 1:2000 . With anti-CYP3A1 we unfortunately cannot see the band of the reference protein and the sample lanes are even without signal or loaded with unspecific staining.
    Looking forward to hearing from you, sincerely

    Read More
    Answer

    Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

    The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

    I would like to reassure you that ab22724 is tested and covered by our 6 month guarantee for use in rat samples and WB. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

    Although the other antibody has worked well using this procedure, individual antibodies will often require optimization. I would like to offer some suggestions to help optimize the results from ab22724. I would also appreciate if you can confirm some details of the procedure.

    1. Please confirm the order number and date of purchase.

    2. Please confirm what the results were. Is it sometimes a clean blot with no bands, and sometimes a lot of bands depending on the dilution of antibody used? I would appreciate if you are able to provide some images which will help me to assess the results. The results from the different dilutions tried.

    3. I can recommend to try using BSA rather than milk to block, at 5%. Changing blocking agent can sometimes help to improve results.

    4. I would appreciate if you are able to describe how the microsomes were prepared?
    I can recommend it would be important to include a whole cell lystate (cells lysed in RIPA buffer) as a positive control.

    5. Have the current vials of secondary antibody been used successfully with other primary antibodies? What were the results of the no primary control?

    6. How was the antibody stored?

    I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

    Read More

    Question

    I'm a researcher interested in your products.
    I need some information on two specific primary antibodies (codes: ab22724 and ab125803). I have to detect the CYP3A family (in particular CYP3A1 and CYP3A2) in a WB experiment using a preparation of rat liver microsomes.
    I see in the datasheets that immunogen of your two products is the same (rat liver cytochrome P450 3A1) and I want to know what of two ab is better for my purpouse and what are the differences between them.
    Thank you for your kindly attention.
    Best regards.

    Read More
    Answer

    Thank you for your email.

    The product ab22724 has been tested with rat samples so it is fully guaranteed. Please check the Abcam abpromise guarantee policy; https://www.abcam.com/index.html?pageconfig=abpromise

    Product ab125803 is now unpublished, which means, it not available for sale anymore.

    I hope this information will be helpful. Should you have any question please do not hesitate to contact me.

    Read More

    Question

    Product code: 22724

    Inquiry: Does this antibody crossreact with rat cyp3A2 in western blot?

    Read More
    Answer

    Thank you for your enquiry.

    I can confirm that ab22724 Cytochrome P450 3A1 antibody [P6] has been tested for cross-reactivity against other CYP3A family members but no evidence was seen to show that it might recognize CYP3A2. I am sorry this has been sourced and tested externally so we regrettably have no data regarding this information from the originating scientist on this occasion.

    I hope this will be helpful. If you have any further questions, please do not hesitate to contact us.

    Read More

    Western blot abreview for Anti-Cytochrome P450 3A1 antibody [P6]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Pig Tissue lysate - other (liver microsomal protein)
    Loading amount
    20 µg
    Specification
    liver microsomal protein
    Gel Running Conditions
    Non-reduced Denaturing
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More

    Dr. Ying Chen

    Verified customer

    Submitted Jun 15 2007

    Western blot abreview for Anti-Cytochrome P450 3A1 antibody [P6]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Rat Tissue lysate - other (liver)
    Loading amount
    20 µg
    Specification
    liver
    Gel Running Conditions
    Non-reduced Denaturing
    Blocking step
    Milk as blocking agent for 14 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Read More

    Dr. Ying Chen

    Verified customer

    Submitted Jun 11 2007

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