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Purified Mouse Anti- Human Topo IIα
Purified Mouse Anti- Human Topo IIα

Western blot analysis of Topo IIα on a HeLa lysate. Lane 1: 1:500, lane 2: 1:1000, lane 3: 1:2000 dilution of the Topo IIα antibody.

Purified Mouse Anti- Human Topo IIα

Immunofluorescent staining of HeLa cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the methanol fix/perm protocol (see Recommended Assay Procedure) and the anti-Topo IIα antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  The image was taken on a Pathway 850 imager using a 20x objective.  This antibody also stained A549 and U2OS cells and can be used with either fix/perm protocol (see Recommended Assay Procedure).

Western blot analysis of Topo IIα on a HeLa lysate. Lane 1: 1:500, lane 2: 1:1000, lane 3: 1:2000 dilution of the Topo IIα antibody.

Immunofluorescent staining of HeLa cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the methanol fix/perm protocol (see Recommended Assay Procedure) and the anti-Topo IIα antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  The image was taken on a Pathway 850 imager using a 20x objective.  This antibody also stained A549 and U2OS cells and can be used with either fix/perm protocol (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing)
Mouse IgG1
Human Topo IIα aa. 1245-1361
Bioimaging, Western blot (Routinely Tested), Immunofluorescence (Tested During Development)
170 kDa
250 µg/ml
AB_398853
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at -20°C.

Recommended Assay Procedures

Methanol Procedure for a 96 well plate:

Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image sample.

Triton-X 100 Procedure for a 96 well plate:

Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 0.1% Triton-X 100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three times with PBS. Image sample.

Product Notices

  1. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Antibody Details
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31/Topo IIa

Eukaryotic DNA topoisomerase II, a ubiquitous ATP-dependent type II topoisomerase, is an essential nuclear enzyme in DNA replication and transcription, chromatin segregation, and cell cycle progression. These enzymes transiently break a pair of complementary strands in double-stranded DNA to form a gate for the passage of duplex DNA. Two isoforms of DNA topoisomerase II have been identified, topo IIα and topo IIß. These have a high degree of homology, except for some divergence in the C-terminal region. Both contain multiple bipartite nuclear localization sequences (NLS) that mediate their subnuclear localization. Topo IIα levels rise during late S phase and peak in G2-M, whereas topo IIß levels remain constant throughout the cell cycle. In addition, topo IIα is expressed in proliferating cells, while topo IIß is expressed in many tissues. The exact role of these two isoforms during cell proliferation is not known, however the differences in cellular expression implicate different physiological roles. Both isoforms may also be important targets for anticancer agents that exert cytotoxicity in proliferating cells via stabilization of a topo II-DNA complex.

This antibody is routinely tested by Western blot analysis and immunofluorescent imaging. Other applications were tested at BD Biosciences Pharmingen during antibody development only.

Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
Citations & References
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Development References (4)

  1. Brown MS, Holden JA, Rahn MP, Perkins SL. Immunohistochemical staining for DNA topoisomerase IIa in Hodgkin's disease. Am J Clin Pathol. 1998; 109(1):39-44. (Biology). View Reference
  2. Herzog CE, Holmes KA, Tuschong LM, Ganapathi R, Zwelling LA. Absence of topoisomerase IIbeta in an amsacrine-resistant human leukemia cell line with mutant topoisomerase IIalpha. Cancer Res. 1998; 58(23):5298-5300. (Biology). View Reference
  3. Mirski SE, Gerlach JH, Cole SP. Sequence determinants of nuclear localization in the alpha and beta isoforms of human topoisomerase II. Exp Cell Res. 1999; 251(2):329-339. (Biology). View Reference
  4. Tsai-Pflugfelder M, Liu LF, Liu AA, et al. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc Natl Acad Sci U S A. 1988; 85(19):7177-7181. (Biology). View Reference
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.