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

1-10 of 20 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - other (top1 cleavage complex purify by CscL gradient)
Gel Running Conditions
Non-reduced Denaturing (Slot Blot)
Loading amount
2 µg
Treatment
DMSO or 10uM CPT
Specification
top1 cleavage complex purify by CscL gradient
Blocking step
Licor Blocking Buffer as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Ms. Cristina Bernal

Verified customer

Submitted Mar 26 2019

Application
Western blot
Sample
Zebrafish Tissue lysate - whole (4 dpf embryos (LWT))
Gel Running Conditions
Reduced Denaturing (4-15 % gradient)
Loading amount
60 µg
Treatment
DMSO 2 h
Specification
4 dpf embryos (LWT)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Dr. Ringaile Zaksauskaite

Verified customer

Submitted Sep 07 2018

Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (U2OS cells)
Total protein in input
500 µg
Immuno-precipitation step
Protein A/G
Specification
U2OS cells

Abcam user community

Verified customer

Submitted Jul 17 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (U2OS Cells)
Gel Running Conditions
Reduced Denaturing (4-12% Gradient Gel)
Loading amount
20 µg
Specification
U2OS Cells
Blocking step
Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jul 14 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Gel Running Conditions
Reduced Denaturing (6%)
Loading amount
50 µg
Specification
HeLa
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 01 2016

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

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

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

Abcam user community

Verified customer

Submitted Sep 19 2014

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.

Read More
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

Mr. Sascha Waidmann

Verified customer

Submitted Sep 05 2012

1-10 of 20 Abreviews or Q&A

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