Overview

  • Product name
    Anti-Topoisomerase I antibody - ChIP Grade
    See all Topoisomerase I primary antibodies
  • Description
    Rabbit polyclonal to Topoisomerase I - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IHC-Fr, WB, IP, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Recombinant fragment, corresponding to amino acids 401 - 600 of Human Topoisomerase I.

  • Positive control
    • HeLa cells.
  • General notes

    This product can be used as part of an assay for sumoylation activity. Human Aos 1 + Uba 2 (ab3804), Ubc 9 (ab3803) and Sumo 1 (ab3801) can be used to promote in vitro sumoylation of a sumoylation marker (human Topoisomerase I protein fragment) (ab3828). The reaction products can be detected using our Sumo 1 (ab3819 and ab3824) and Topoisomerase I (ab3825) antibodies. Sumoylation assays are carried out in a final volume of 20µl in reaction conditions (20 mM Hepes pH 7.5, 5mM MgCl2, 2mM ATP). This antibody is equivalent to the antibody used in our Sumoylation Control kits. Sumoylation Protocol: 1. Prepare a suitable purified substrate protein. (For the control, use 2µl Topoisomerase I marker for each reaction.) 2. In each reaction, add 4µl E2 to substrate first, then 2µl Sumo 1, 2µl 10x reaction buffer, 2µl E1. Finally, add H2O to bring up to 20µl. We would recommend adding fresh 2mM ATP to be sure that sufficient energy is supplied. 3. The best reaction concentration of proteins is as following: Aos 1 + Uba 2: 7.5µg/ml. Ubc 9: 50µg/ml. SUMO 1: 50µg/ml. For the control assay we recommend running the assay at 37ºC for 30-60 minutes. 4. Detect the reaction products by Western blot using a suitable antibody. For the control reaction use 1/1000 dilution of the supplied Topoisomerase I antibody. Four sumoylated bands should be seen on the gel for the control reaction. This assay has been shown to work with crude extracts. Be aware that Uba 2 contains his-rich regions which might cross-react with antibodies against the 6x-His epitope tag. During western analysis with anti-6x-His antibodies, Uba 2 at 80 kDa might be shown.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Purity
    Whole antiserum
  • Primary antibody notes
    This product can be used as part of an assay for sumoylation activity. Human Aos 1 + Uba 2 (ab3804), Ubc 9 (ab3803) and Sumo 1 (ab3801) can be used to promote in vitro sumoylation of a sumoylation marker (human Topoisomerase I protein fragment) (ab3828). The reaction products can be detected using our Sumo 1 (ab3819 and ab3824) and Topoisomerase I (ab3825) antibodies. Sumoylation assays are carried out in a final volume of 20µl in reaction conditions (20 mM Hepes pH 7.5, 5mM MgCl2, 2mM ATP). This antibody is equivalent to the antibody used in our Sumoylation Control kits. Sumoylation Protocol: 1. Prepare a suitable purified substrate protein. (For the control, use 2µl Topoisomerase I marker for each reaction.) 2. In each reaction, add 4µl E2 to substrate first, then 2µl Sumo 1, 2µl 10x reaction buffer, 2µl E1. Finally, add H2O to bring up to 20µl. We would recommend adding fresh 2mM ATP to be sure that sufficient energy is supplied. 3. The best reaction concentration of proteins is as following: Aos 1 + Uba 2: 7.5µg/ml. Ubc 9: 50µg/ml. SUMO 1: 50µg/ml. For the control assay we recommend running the assay at 37ºC for 30-60 minutes. 4. Detect the reaction products by Western blot using a suitable antibody. For the control reaction use 1/1000 dilution of the supplied Topoisomerase I antibody. Four sumoylated bands should be seen on the gel for the control reaction. This assay has been shown to work with crude extracts. Be aware that Uba 2 contains his-rich regions which might cross-react with antibodies against the 6x-His epitope tag. During western analysis with anti-6x-His antibodies, Uba 2 at 80 kDa might be shown.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab3825 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
ICC/IF Use at an assay dependent concentration. PubMed: 20655466
IHC-Fr Use at an assay dependent concentration.
WB 1/500 - 1/1000. Detects a band of approximately 92 kDa (predicted molecular weight: 91 kDa).
IP Use at an assay dependent concentration. PubMed: 17290216
ChIP Use at an assay dependent concentration. PubMed: 20655466

Target

  • Function
    The reaction catalyzed by topoisomerases leads to the conversion of one topological isomer of DNA to another.
  • Involvement in disease
    Note=A chromosomal aberration involving TOP1 is found in a form of therapy-related myelodysplastic syndrome. Translocation t(11;20)(p15;q11) with NUP98.
  • Sequence similarities
    Belongs to the eukaryotic type I topoisomerase family.
  • Post-translational
    modifications
    Sumoylated. Lys-117 is the main site of sumoylation. Sumoylation plays a role in partitioning TOP1 between nucleoli and nucleoplasm. Levels are dramatically increased on camptothecin (CPT) treatment.
  • Cellular localization
    Nucleus > nucleolus. Nucleus > nucleoplasm. Diffuse nuclear localization with some enrichment in nucleoli. On CPT treatment, cleared from nucleoli into nucleoplasm. Sumolyated forms found in both nucleoplasm and nucleoli.
  • Information by UniProt
  • Database links
  • Alternative names
    • DNA topoisomerase 1 antibody
    • DNA topoisomerase I antibody
    • NUP98 fusion gene antibody
    • TOP 1 antibody
    • TOP I antibody
    • TOP1 antibody
    • TOP1_HUMAN antibody
    • TOPI antibody
    • Topoisomerase (DNA) I antibody
    • Topoisomerase 1 antibody
    • Topoisomerase1 antibody
    • TopoisomeraseI antibody
    • Type I DNA topoisomerase antibody
    see all

Images

  • ab3825 staining Topoisomerase I in human 293T cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 3% BSA for 1 hour at 22°C. Samples were incubated with primary antibody (1/100 in PBS + 1% BSA) for 1 hour at 22°C. An Alexa Fluor® 488-conjugated donkey anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.

    See Abreview

  • blue: DAPI; green: Topo I. Hela cell staining.
  • Primary antibody incubated at a 1/1000 dilution for 60 minutes at 25ºC.

  • All lanes : Anti-Topoisomerase I antibody - ChIP Grade (ab3825) at 1/5000 dilution

    Lane 1 : SW210.5 human SCLC cell xenograft lysate.
    Lane 2 : 5M2 human NSCLC cell lysate (bicarb. buffer).
    Lane 3 : 5M2 human NSCLC cell lysate (M-Per buffer).
    Lane 4 : Normal human lung tissue lysate.
    Lane 5 : A431 cell (human epidermoid carcinoma) lysate .
    Lane 6 : Rh30 cell (human rhabdomyosarcoma) lysate.

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti rabbit heavy and light (HRP) at 1/25000 dilution

    Performed under reducing conditions.

    Predicted band size: 91 kDa



    This image is courtesy of an Abreview submitted by Mike Campa on 7 September 2005. We do not have any further information relating to this image.

    See Abreview

  • All lanes : Anti-Topoisomerase I antibody - ChIP Grade (ab3825) at 1/200 dilution

    Lane 1 : K562 nuclear lysate
    Lane 2 : Mouse N2a whole cell lysate at 100 µg

    Secondary
    All lanes : Donkey anti-rabbit IgG HRP polyclonal at 1/1000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 91 kDa
    Observed band size: 100 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 35 kDa (possible non-specific binding)


    Exposure time: 15 seconds

    See Abreview

References

This product has been referenced in:
  • Ahmad M  et al. Topoisomerase 3ß is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction. Nucleic Acids Res 45:2704-2713 (2017). Read more (PubMed: 28039324) »
  • Bansal K  et al. The transcriptional regulator Aire binds to and activates super-enhancers. Nat Immunol 18:263-273 (2017). Read more (PubMed: 28135252) »
See all 20 Publications for this product

Customer reviews and Q&As

1-9 of 9 Q&A

Answer

Thank you for contacting us. Because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

If the product is to be used in an untested species or application, you may be eligible for our AbTrial program if the antibody has not yet been purchased. Please contact our Scientific Support team by replying to this email prior to purchase for more information. Or you can find more information about this program at the following link:

www.abcam.com/abtrial

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

https://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

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Answer


I am very pleased to hear you would like to accept our offer and test ab3825 in Arabidopsis. This code will give you: 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview forArabidopsis and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.


Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Question
Answer

Thank you very much for your interest in ab3825. We unfortunately do not have test samples, however we are happy to make you an interesting testing offer.
Indeed, ab3825 has not been tested on Arabidopsis samples. According to the alignment of the immungen sequence (human sequence from amino acid 400 to 600), it could be possible, however it needs experimental testing to be sure. Please see here for the alignment: http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20120823-115053-0089-8826671-pg

I can therefore make the following offer: We can offer a discount off a future purchase if you buy ab3825 now, test it inArabidopsis and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of: 1 free primary antibody.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab3825 in Arabidopsis. I will then send a discount code. This code must be issued before purchasing ab3825 so please wait for my reply before ordering.

2. Purchase ab3825 either by phone, fax, or online (www.abcam.com).

3. Test it in Arabidopsis.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for anyprimary antibodyordered and the discount code is valid for 4 months after issue.
Please remember that submission of the Abreview is sufficient for the discount code to become active.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab3825 turns out to be unsuitable for Arabidopsis, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

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Question
Answer

Thank you for contacting us.

To our knowledge, reactivity of the antibody ab3825 with Arabidopsis Top1a has never been tested.

To check the probability of cross-reaction, I aligned the amino acid sequence of Arabidopsis Top1a (found at this link: http://www.uniprot.org/uniprot/P30181) with the sequence of the immunogen of the antibody, which is the fragment from amino acids 401 - 600 of human Top1a (found at this link: http://www.uniprot.org/uniprot/P11387)

The result is that there is only 53% overall identity, but also some regions of 10- 20 amino acids that are completely conserved.

In short, the antibody may recognize Arabidopsis Top1a, if it recognizes one or more of the regions that are conserved. The antibody is polyclonal, that is, it recognizes multiple epitopes, but the locations of the epitopes are unknown.

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

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Answer

Thank you for your email.

I've added the discount code to your review, so it is now active and can be redeemed for a free primary antibody. Thank you for your review!

Please let me know if you need anything else. Have a great day!

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Question
Answer

Thank you for your call today. I've issued you the discount code below, and let me know if you have any questions or if there is anything else that we can do for you.

DISCOUNT CODE: ****
Expiration date: August 11, 2012

This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview forab3825 in mouse samplesand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Answer

Thank you for your enquiry. The ammonium sulfate-precipitated anti-topoisomerase I antiserum is kept in PBS with 1% BSA and then lyophilized. Lyophilized ab3825 antibody should be reconstituted with 100 ul of pure H2O before use. Please let me know if you need further assistance.

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Answer

Thank you for your enquiry. All the information that we have regarding ab3825 and its application in immunofluorescence is located on the online datasheet. If you have any more questions, please contact us again.

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Answer

Please refer to the Abcam protocol for frozen sections on teh Abcam homepage.

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