Anti-Topoisomerase I antibody - ChIP Grade (ab3825)

Overview

  • Product nameAnti-Topoisomerase I antibody - ChIP Grade
    See all Topoisomerase I primary antibodies
  • Description
    Rabbit polyclonal to Topoisomerase I - ChIP Grade
  • Tested applicationsSuitable 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

  • FormLiquid
  • Storage instructionsShipped 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.
  • PurityWhole antiserum
  • Primary antibody notesThis 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.
  • ClonalityPolyclonal
  • IsotypeIgG
  • 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

  • FunctionThe reaction catalyzed by topoisomerases leads to the conversion of one topological isomer of DNA to another.
  • Involvement in diseaseNote=A chromosomal aberration involving TOP1 is found in a form of therapy-related myelodysplastic syndrome. Translocation t(11;20)(p15;q11) with NUP98.
  • Sequence similaritiesBelongs 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 localizationNucleus > 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

Anti-Topoisomerase I antibody - ChIP Grade 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.


  • Predicted band size : 91 kDa
    Primary antibody incubated at a 1/1000 dilution for 60 minutes at 25ºC. 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
    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
    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

    This image is courtesy of an anonymous Abreview

    See Abreview

References for Anti-Topoisomerase I antibody - ChIP Grade (ab3825)

This product has been referenced in:
  • Bhatia V  et al. BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2. Nature 511:362-5 (2014). Read more (PubMed: 24896180) »
  • Naughton C  et al. Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures. Nat Struct Mol Biol 20:387-95 (2013). ChIP . Read more (PubMed: 23416946) »

See all 10 Publications for this product

Product Wall

Application Western blot
Loading amount 100000 cells
Gel Running Conditions Reduced Denaturing (10%)
Sample Chinese Hamster Cell lysate - whole cell (EM9)
Specification EM9
Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Abcam user community

Verified customer

Submitted Nov 18 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12% SDS-PAGE)
Sample Human Cell lysate - whole cell (293T)
Specification 293T
Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Abcam user community

Verified customer

Submitted Sep 19 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 22°C
Sample Human Cell (293T)
Specification 293T
Permeabilization Yes - 0.5% Triton X100
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 19 2014

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 guaran...

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Abcam has not validated the combination of species/application used in this Abreview.
Application Western blot
Sample Arabidopsis thaliana Tissue lysate - nuclear (Seedlings)
Loading amount 50 µg
Specification Seedlings
Gel Running Conditions Reduced Denaturing (8% SDS PAGE)
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Mr. Sascha Waidmann

Verified customer

Submitted Sep 05 2012


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 th...

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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...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Cell lysate - whole cell (Neuro-2A)
Loading amount 100 µg
Specification Neuro-2A
Gel Running Conditions Reduced Denaturing (7.5%)
Blocking step StartingBlock T20 (TBS) Blocking Buffer as blocking agent for 20 minute(s) · Concentration: 100% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted May 02 2012

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 (fou...

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