ARNT Colorimetric Cell-Based ELISA

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SKU:
CBCAB00379
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
Frequently bought together:

Description

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ARNT Colorimetric Cell-Based ELISA

The ARNT Colorimetric Cell-Based ELISA Kit is a powerful tool for studying the activity of ARNT (Aryl hydrocarbon receptor nuclear translocator) in cell cultures. This kit allows for the precise and quantitative measurement of ARNT levels in a simple and efficient manner.ARNT is a key transcription factor that plays a crucial role in the regulation of genes involved in various biological processes, including xenobiotic metabolism, cell growth, and differentiation. Dysregulation of ARNT has been implicated in a variety of diseases, making it a valuable target for research and drug development.

With high sensitivity and specificity, this kit provides accurate and reliable results, allowing researchers to investigate the role of ARNT in cellular pathways and disease mechanisms. Whether studying cancer, metabolic disorders, or environmental toxins, the ARNT Colorimetric Cell-Based ELISA Kit offers a versatile and robust solution for your research needs.

Product Name:ARNT Colorimetric Cell-Based ELISA
Product Code:CBCAB00379
ELISA Type:Cell-Based
Target:ARNT
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of ARNT concentration is achieved by an indirect ELISA format. In essence, ARNT is captured by ARNT-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: 405, UniProt ID: P27540, OMIM: 126110, Unigene: Hs.632446
Gene Symbol:ARNT
Sub Type:None
UniProt Protein Function:Required for activity of the Ah (dioxin) receptor. This protein is required for the ligand-binding subunit to translocate from the cytosol to the nucleus after ligand binding. The complex then initiates transcription of genes involved in the activation of PAH procarcinogens. The heterodimer with HIF1A or EPAS1/HIF2A functions as a transcriptional regulator of the adaptive response to hypoxia.
NCBI Summary:This gene encodes a protein containing a basic helix-loop-helix domain and two characteristic PAS domains along with a PAC domain. The encoded protein binds to ligand-bound aryl hydrocarbon receptor and aids in the movement of this complex to the nucleus, where it promotes the expression of genes involved in xenobiotic metabolism. This protein is also a co-factor for transcriptional regulation by hypoxia-inducible factor 1. Chromosomal translocation of this locus with the ETV6 (ets variant 6) gene on chromosome 12 have been described in leukemias. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Oct 2013]
UniProt Code:P27540
NCBI GenInfo Identifier:114163
NCBI Gene ID:405
NCBI Accession:P27540.1
UniProt Secondary Accession:P27540,Q59ED4, Q5QP39, Q8NDC7, B2R9H1, C4AMA1, F8WAP6
UniProt Related Accession:P27540
Molecular Weight:86,366 Da
NCBI Full Name:Aryl hydrocarbon receptor nuclear translocator
NCBI Synonym Full Names:aryl hydrocarbon receptor nuclear translocator
NCBI Official Symbol:ARNT  
NCBI Official Synonym Symbols:HIF1B; TANGO; bHLHe2; HIF1BETA; HIF-1beta; HIF1-beta; HIF-1-beta  
NCBI Protein Information:aryl hydrocarbon receptor nuclear translocator
UniProt Protein Name:Aryl hydrocarbon receptor nuclear translocator
UniProt Synonym Protein Names:Class E basic helix-loop-helix protein 2; bHLHe2; Dioxin receptor, nuclear translocator; Hypoxia-inducible factor 1-beta; HIF-1-beta; HIF1-beta
Protein Family:Aryl hydrocarbon receptor nuclear translocator
UniProt Gene Name:ARNT  
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-ARNT 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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