Anti-Topoisomerase I antibody (ab3825)
Key features and details
- Rabbit polyclonal to Topoisomerase I
- Suitable for: WB, ICC
- Reacts with: Human
- Isotype: IgG
Overview
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Product name
Anti-Topoisomerase I antibody
See all Topoisomerase I primary antibodies -
Description
Rabbit polyclonal to Topoisomerase I -
Host species
Rabbit -
Tested applications
Suitable for: WB, ICCmore details -
Species reactivity
Reacts with: Human -
Immunogen
Recombinant fragment corresponding to Human Topoisomerase I aa 401-600.
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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
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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. -
Storage buffer
Constituent: 100% PBS -
Concentration information loading... -
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
Images
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Immunocytochemistry - Anti-Topoisomerase I antibody (ab3825) This image is courtesy of an anonymous Abreview
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.
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Immunocytochemistry - Anti-Topoisomerase I antibody (ab3825)blue: DAPI; green: Topo I. Hela cell staining. -
Western blot - Anti-Topoisomerase I antibody (ab3825)Primary antibody incubated at a 1/1000 dilution for 60 minutes at 25ºC.
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Western blot - Anti-Topoisomerase I antibody (ab3825)All lanes : Anti-Topoisomerase I antibody (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. -
Western blot - Anti-Topoisomerase I antibody (ab3825) This image is courtesy of an anonymous AbreviewAll lanes : Anti-Topoisomerase I antibody (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
