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TOP2B Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00202
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

TOP2B Colorimetric Cell-Based ELISA Kit

The TOP2B Colorimetric Cell-Based ELISA Kit is a powerful tool for scientists and researchers looking to accurately assess the activity of topoisomerase II beta (TOP2B) in cell samples. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.TOP2B is an important enzyme involved in DNA replication and gene transcription, making it a key player in cell growth and division. Dysregulation of TOP2B activity has been linked to various diseases, including cancer and neurological disorders, highlighting the significance of studying this enzyme in research.

With the TOP2B Colorimetric Cell-Based ELISA Kit, researchers can easily measure TOP2B activity in cell lysates and culture supernatants, allowing for a better understanding of its role in cellular processes. This kit is simple to use and provides accurate quantification of TOP2B activity, making it an essential tool for studying cell biology and identifying potential therapeutic targets.

Product Name:TOP2B Colorimetric Cell-Based ELISA
Product Code:CBCAB00202
ELISA Type:Cell-Based
Target:TOP2B
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The TOP2B Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TOP2B protein expression profile in cells. The kit can be used for measuring the relative amounts of TOP2B in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TOP2B.

Qualitative determination of TOP2B concentration is achieved by an indirect ELISA format. In essence, TOP2B is captured by TOP2B-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 7155, UniProt ID: Q02880, OMIM: 126431, Unigene: Hs.475733
Gene Symbol:TOP2B
Sub Type:None
UniProt Protein Function:Control of topological states of DNA by transient breakage and subsequent rejoining of DNA strands. Topoisomerase II makes double-strand breaks.
NCBI Summary:This gene encodes a DNA topoisomerase, an enzyme that controls and alters the topologic states of DNA during transcription. This nuclear enzyme is involved in processes such as chromosome condensation, chromatid separation, and the relief of torsional stress that occurs during DNA transcription and replication. It catalyzes the transient breaking and rejoining of two strands of duplex DNA which allows the strands to pass through one another, thus altering the topology of DNA. Two forms of this enzyme exist as likely products of a gene duplication event. The gene encoding this form, beta, is localized to chromosome 3 and the alpha form is localized to chromosome 17. The gene encoding this enzyme functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. Reduced activity of this enzyme may also play a role in ataxia-telangiectasia. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Aug 2016]
UniProt Code:Q02880
NCBI GenInfo Identifier:339810
NCBI Gene ID:7155
NCBI Accession:AAA61210.1
UniProt Secondary Accession:Q02880,Q13600, Q9UMG8, Q9UQP8,
UniProt Related Accession:Q02880
Molecular Weight:182,663 Da
NCBI Full Name:topoisomerase II, partial
NCBI Synonym Full Names:DNA topoisomerase II beta
NCBI Official Symbol:TOP2B  
NCBI Official Synonym Symbols:TOPIIB; top2beta  
NCBI Protein Information:DNA topoisomerase 2-beta
UniProt Protein Name:DNA topoisomerase 2-beta
UniProt Synonym Protein Names:DNA topoisomerase II, beta isozyme
Protein Family:DNA topoisomerase
UniProt Gene Name:TOP2B  
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies (Anti-TOP2B Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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