Topoisomerase I Antibody F14
(Catalog # AP006)

Specificity: Rabbit polyclonal antibody against human topoisomerase I.

Antigen used for immunizations:
Prokaryotic recombinant protein corresponding to the amino acid residues 1 to 200 of the human topoisomerase I [access no: P11387] molecule.

Storage Buffer and Reconstitution:
The ammonium sulfate-precipitated anti-topoisomerase I antiserum is kept in PBS and then lyophilized. Lyophilized F14 antybody should be reconstituted with 100 ul of H₂O before use.

Recommendations on use:

Immunofluorescence:

Typical working dilution on frozen sections 1:50-1:100. 60 minutes primary antibody incubates at 25 ^oC (as shown in Fig. 1).

Western blotting: Recommended:

Working dilution on Western blotting 1:1000. 60 minutes primary antibody incubates at 25 ^oC (as shown in Fig. 2).

Fig. 1: blue: DAPI; green: Topo I                   Fig. 2

Positive controls: HeLa cells

Staining pattern: Nuclear

Storage and stability: Store unopened antibody at 4 ^oC or lower. Under these conditions, there is no significant loss in product performance up to one year from the purchase date. The reconstituted antibody is stable for at least two months when stored at 4 ^oC. For long term storage, it is recommended that aliquots of the antibody are frozen at
-20 ^oC (frost-free freezers are not recommended). Repeated freezing and thawing must be avoided. Prepare working dilutions on the day of use.

Legal consideration: FOR RESEARCH USE ONLY.

Application: 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 re-ligated 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. In normal eukaryotic cells, DNA topoisomerase I does not show relevant fluctuations across the cell cycle, unlike DNA topoisomerase II alpha. 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 ³²P transfer assays, have been demonstrated in colorectal tumors compared with normal colon mucosa as a result of increased transcription or mRNA stability. Renal tumors were found not to show any differential levels compared with normal kidney. Topo I-F14 antibody may be useful for the evaluation of DNA topoisomerase I expression in normal tissues, solid tumors and in further studies of ovarian, colorectal, cervical and prostatic tumors.

References:

Holden J A, Rahn M P, Jolles C J, et al.. Immunohistochemical detection of DNA topoisomerase I in formalin fixed, paraffin wax embedded normal tissues and in ovarian carcinomas. J Clin Pathol: Mol Pathol. 50: 247-253 (1997).

McLeod H L, Douglas F, Oates M, et al.. Topoisomerase I and II activity in human breast, cervix, lung and colon cancer. Int J Cancer. 59: 607-611 (1994).

Shero J H, Bordwell B, Rothfield N F, et al.. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science. 231: 737-740 (1986).