Topoisomerase I antibody (ab3825)

Product Name 

Topoisomerase I antibody

Product type 

Primary antibodies

Description 

Rabbit polyclonal to Topoisomerase I

Immunogen 

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

Reacts with 
(species key)

Hu

Tested applications 
(see key)

ICC/IF, IHC-Fr, IP, WB

Abreviews 

Customer reviews (see key)

Read about Abreviews (Read all Abreviews)

Application notes 
(see key)

Recommended dilutions
Immunofluorescence: 1/200-1/500 on frozen sections. Incubate for 60 minutes at 25ºC.
IHC-Fr: Use at an assay dependent dilution.
IP: Use at an assay dependent dilution from (PubMed:17290216).
WB: 1/1000. Detects a band of approximately 92 kDa (predicted molecular weight: 91 kDa). Incubate for 60 minutes at 25ºC.

Not tested in other applications.

Optimal dilutions / concentrations should be determined by the end user.

Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.

Positive control 
(see definition)

HeLa cells.

Cellular localization 

Cytoplasmic and Nuclear.

Research areas 

Cancer >> Drug resistance >> Topoisomerases
Chromatin and Nuclear Signaling >> DNA / RNA >> DNA Synthesis >> Topoisomerases

Relevance 

Topoisomerases are nuclear enzymes involved in a variety of cellular activities such as chromosome condensation, DNA replication, transcription, recombination and segregation at mitosis. Human topoisomerase I is a 100kD protein capable of relaxing positively and negatively supercoiled DNA by performing a transient single stranded nick which is then religated at the end of the reaction. It has been shown that the enzyme is located in regions of the genome that are undergoing active RNA synthesis, where it probably reduces superhelical stresses in the DNA, enabling RNA polymerase to function properly. Both DNA topoisomerases I and II have been found to be targets of autoantibodies in the sera of patients with certain autoimmune diseases such as systemic lupus erythematosus and also of some anti tumor drugs and antibiotics. Elevated levels of DNA topoisomerase I, detected by transfer assays, have been demonstrated in colorectal tumors compared with normal colon mucosa as a result of increased transcription or mRNA stability.

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.

Database links 

Raised in 

Rabbit

Clonality 

Polyclonal

Isotype 

IgG

Purity 

Whole antiserum

Storage buffer 

n/a

Form 

Liquid

Concentration 

Concentration not determined - why is this?

Storage instructions 

Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.

See below for Topoisomerase I antibody images, references, products related to ab3825 and other tools.

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References for Topoisomerase I antibody (ab3825)

  This product has been used in: (two most recent references)

Abdurashidova G et al. Functional interactions of DNA topoisomerases with a human replication origin. EMBO J 26:998-1009 (2007). PubMed: 17290216

Abdurashidova G et al. Functional interactions of DNA topoisomerases with a human replication origin. EMBO J 26:998-1009 (2007). PubMed: 17290216

See all 3 publication references for this product.


If you publish research using ab3825 please let us know so that we can cite the reference on this datasheet.

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