Androgen Receptor (Phospho-Tyr363) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00498
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
Frequently bought together:

Description

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Androgen Receptor (Phospho-Tyr363)Colorimetric Cell-Based ELISA Kit

The Androgen Receptor Phospho-Tyr363 Colorimetric Cell-Based ELISA Kit is designed for the accurate detection of phosphorylated tyrosine 363 levels in cell lysates and tissue samples. This kit features high sensitivity and specificity, ensuring reliable and reproducible results, making it ideal for studying the role of androgen receptor phosphorylation in various cellular processes.The phosphorylation of tyrosine 363 on the androgen receptor is a critical post-translational modification that regulates its activity and function in signaling pathways involved in growth, development, and disease progression.

Dysregulation of this phosphorylation event has been implicated in various cancers, including prostate cancer, making this kit a valuable tool for researchers studying the mechanisms of androgen receptor signaling in cancer and other diseases.Overall, the Androgen Receptor Phospho-Tyr363 Colorimetric Cell-Based ELISA Kit is essential for advancing our understanding of androgen receptor biology and its potential role as a therapeutic target in cancer and other diseases.

Product Name:Androgen Receptor (Phospho-Tyr363) Colorimetric Cell-Based ELISA
Product Code:CBCAB00498
ELISA Type:Cell-Based
Target:Androgen Receptor (Phospho-Tyr363)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Androgen Receptor (Phospho-Tyr363) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Androgen Receptor protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Androgen Receptor in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Androgen Receptor phosphorylation.

Qualitative determination of Androgen Receptor (Phospho-Tyr363) concentration is achieved by an indirect ELISA format. In essence, Androgen Receptor (Phospho-Tyr363) is captured by Androgen Receptor (Phospho-Tyr363)-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: 367, UniProt ID: P10275, OMIM: 109200/300068/312300/313200/313700, Unigene: Hs.496240
Gene Symbol:AR
Sub Type:Phospho
UniProt Protein Function:AR: a nuclear hormone receptor and transcription factor. Regulates gene expression and affects cellular proliferation and differentiation in target tissues. Two splice-variant isoforms have been described.
UniProt Protein Details:

Protein type:Transcription factor; Nuclear receptor; DNA-binding

Chromosomal Location of Human Ortholog: Xq12

Cellular Component: nucleoplasm; protein complex; cytoplasm; nuclear chromatin; nucleus

Molecular Function:protein dimerization activity; protein binding; ligand-dependent nuclear receptor activity; androgen receptor activity; enzyme binding; DNA binding; androgen binding; zinc ion binding; beta-catenin binding; chromatin binding; transcription factor binding; transcription factor activity; receptor binding

Biological Process: prostate gland development; transcription initiation from RNA polymerase II promoter; intracellular receptor-mediated signaling pathway; transcription, DNA-dependent; positive regulation of transcription, DNA-dependent; signal transduction; protein oligomerization; activation of NF-kappaB transcription factor; negative regulation of integrin biosynthetic process; cell proliferation; cell-cell signaling; transport; androgen receptor signaling pathway; positive regulation of cell proliferation; positive regulation of transcription from RNA polymerase III promoter; gene expression; steroid hormone mediated signaling; positive regulation of transcription from RNA polymerase II promoter; positive regulation of integrin biosynthetic process; cell growth; sex differentiation; positive regulation of phosphorylation

Disease: Androgen Insensitivity Syndrome; Prostate Cancer; Androgen Insensitivity, Partial; Hypospadias 1, X-linked; Spinal And Bulbar Muscular Atrophy, X-linked 1

NCBI Summary:The androgen receptor gene is more than 90 kb long and codes for a protein that has 3 major functional domains: the N-terminal domain, DNA-binding domain, and androgen-binding domain. The protein functions as a steroid-hormone activated transcription factor. Upon binding the hormone ligand, the receptor dissociates from accessory proteins, translocates into the nucleus, dimerizes, and then stimulates transcription of androgen responsive genes. This gene contains 2 polymorphic trinucleotide repeat segments that encode polyglutamine and polyglycine tracts in the N-terminal transactivation domain of its protein. Expansion of the polyglutamine tract causes spinal bulbar muscular atrophy (Kennedy disease). Mutations in this gene are also associated with complete androgen insensitivity (CAIS). Two alternatively spliced variants encoding distinct isoforms have been described. [provided by RefSeq, Jul 2008]
UniProt Code:P10275
NCBI GenInfo Identifier:113830
NCBI Gene ID:367
NCBI Accession:P10275.2
UniProt Secondary Accession:P10275,Q9UD95, A2RUN2, B1AKD7,
UniProt Related Accession:P10275
Molecular Weight:44,643 Da
NCBI Full Name:Androgen receptor
NCBI Synonym Full Names:androgen receptor
NCBI Official Symbol:AR  
NCBI Official Synonym Symbols:KD; AIS; TFM; DHTR; SBMA; HYSP1; NR3C4; SMAX1; HUMARA  
NCBI Protein Information:androgen receptor; androgen nuclear receptor variant 2; dihydrotestosterone receptor; nuclear receptor subfamily 3 group C member 4
UniProt Protein Name:Androgen receptor
UniProt Synonym Protein Names:Dihydrotestosterone receptor; Nuclear receptor subfamily 3 group C member 4
Protein Family:Allatostatin
UniProt Gene Name:AR  
UniProt Entry Name:ANDR_HUMAN
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-Androgen Receptor (Phospho-Tyr363) Antibody, Anti-Androgen Receptor 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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