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Retinoid X Receptor gamma Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00844
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS
Product Name:Retinoid X Receptor gamma Colorimetric Cell-Based ELISA
Product Code:CBCAB00844
ELISA Type:Cell-Based
Target:Retinoid X Receptor gamma
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of Retinoid X Receptor gamma concentration is achieved by an indirect ELISA format. In essence, Retinoid X Receptor gamma is captured by Retinoid X Receptor gamma-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: 6258, UniProt ID: P48443, OMIM: 180247, Unigene: Hs.26550
Gene Symbol:RXRG
Sub Type:None
UniProt Protein Function:RXRG: Receptor for retinoic acid. Retinoic acid receptors bind as heterodimers to their target response elements in response to their ligands, all-trans or 9-cis retinoic acid, and regulate gene expression in various biological processes. The RAR/RXR heterodimers bind to the retinoic acid response elements (RARE) composed of tandem 5'-AGGTCA-3' sites known as DR1-DR5. The high affinity ligand for RXRs is 9-cis retinoic acid. Homodimer. Heterodimer with a RAR molecule. Binds DNA preferentially as a RAR/RXR heterodimer. Belongs to the nuclear hormone receptor family. NR2 subfamily.
UniProt Protein Details:

Protein type:DNA-binding; Nuclear receptor

Chromosomal Location of Human Ortholog: 1q22-q23

Cellular Component: nucleoplasm

Molecular Function:protein binding; zinc ion binding; retinoid-X receptor activity; steroid hormone receptor activity

Biological Process: retinoic acid receptor signaling pathway; neuron differentiation; transcription initiation from RNA polymerase II promoter; skeletal muscle development; response to retinoic acid; regulation of myelination; heart development; steroid hormone mediated signaling; gene expression; transmembrane transport; protein homotetramerization; peripheral nervous system development

NCBI Summary:This gene encodes a member of the retinoid X receptor (RXR) family of nuclear receptors which are involved in mediating the antiproliferative effects of retinoic acid (RA). This receptor forms dimers with the retinoic acid, thyroid hormone, and vitamin D receptors, increasing both DNA binding and transcriptional function on their respective response elements. This gene is expressed at significantly lower levels in non-small cell lung cancer cells. Alternatively spliced transcript variants have been described. [provided by RefSeq, Jun 2010]
UniProt Code:P48443
NCBI GenInfo Identifier:1350913
NCBI Gene ID:6258
NCBI Accession:P48443.1
UniProt Secondary Accession:P48443,Q6IBU7, A6NIP1,
UniProt Related Accession:P48443
Molecular Weight:50,871 Da
NCBI Full Name:Retinoic acid receptor RXR-gamma
NCBI Synonym Full Names:retinoid X receptor, gamma
NCBI Official Symbol:RXRG  
NCBI Official Synonym Symbols:RXRC; NR2B3  
NCBI Protein Information:retinoic acid receptor RXR-gamma; nuclear receptor subfamily 2 group B member 3
UniProt Protein Name:Retinoic acid receptor RXR-gamma
UniProt Synonym Protein Names:Nuclear receptor subfamily 2 group B member 3; Retinoid X receptor gamma
UniProt Gene Name:RXRG  
UniProt Entry Name:RXRG_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-Retinoid X Receptor gamma 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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