Epigenetics and Nuclear Signaling
NR1I3 Colorimetric Cell-Based ELISA
- SKU:
- CBCAB01101
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Epigenetics and Nuclear Signaling
- Reactivity:
- Human
- Detection Method:
- Colorimetric
Description
| Product Name: | NR1I3 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01101 |
| ELISA Type: | Cell-Based |
| Target: | NR1I3 |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The NR1I3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NR1I3 protein expression profile in cells. The kit can be used for measuring the relative amounts of NR1I3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NR1I3.
Qualitative determination of NR1I3 concentration is achieved by an indirect ELISA format. In essence, NR1I3 is captured by NR1I3-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: 9970, UniProt ID: Q14994, OMIM: 603881, Unigene: Hs.349642 |
| Gene Symbol: | NR1I3 |
| Sub Type: | None |
| UniProt Protein Function: | NR1I3: Binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes. Transactivates both the phenobarbital responsive element module of the human CYP2B6 gene and the CYP3A4 xenobiotic response element. Interacts with ECT2. Heterodimer of NR1I3 and RXR. Interacts with PSMC4. Directly interacts with DNAJC7. The DNAJC7-NR1I3 complex may also include HSP90. By dexamethasone. Predominantly expressed in liver. Belongs to the nuclear hormone receptor family. NR1 subfamily. 7 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Nuclear receptor; DNA-binding Chromosomal Location of Human Ortholog: 1q23.3 Cellular Component: nucleoplasm; cytoskeleton; cytoplasm; cytosol; nucleus Molecular Function:ligand-dependent nuclear receptor activity; androgen receptor activity; DNA binding; zinc ion binding; transcription coactivator activity; thyroid hormone receptor activity; transcription factor activity Biological Process: transcription initiation from RNA polymerase II promoter; intracellular receptor-mediated signaling pathway; androgen receptor signaling pathway; gene expression; steroid hormone mediated signaling; positive regulation of transcription from RNA polymerase II promoter; signal transduction; negative regulation of transcription, DNA-dependent |
| NCBI Summary: | This gene encodes a member of the nuclear receptor superfamily, and is a key regulator of xenobiotic and endobiotic metabolism. The protein binds to DNA as a monomer or a heterodimer with the retinoid X receptor and regulates the transcription of target genes involved in drug metabolism and bilirubin clearance, such as cytochrome P450 family members. Unlike most nuclear receptors, this transcriptional regulator is constitutively active in the absence of ligand but is regulated by both agonists and inverse agonists. Ligand binding results in translocation of this protein to the nucleus, where it activates or represses target gene transcription. These ligands include bilirubin, a variety of foreign compounds, steroid hormones, and prescription drugs. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q14994 |
| NCBI GenInfo Identifier: | 49066046 |
| NCBI Gene ID: | 9970 |
| NCBI Accession: | Q14994.2 |
| UniProt Secondary Accession: | Q14994,Q0VAC9, Q4U0F0, E9PB75, E9PC13, E9PDU3, E9PGH6 E9PH10, E9PHC8, E9PHN4, F1D8Q0, F1D8Q1, |
| UniProt Related Accession: | Q14994 |
| Molecular Weight: | 352 |
| NCBI Full Name: | Nuclear receptor subfamily 1 group I member 3 |
| NCBI Synonym Full Names: | nuclear receptor subfamily 1, group I, member 3 |
| NCBI Official Symbol: | NR1I3 |
| NCBI Official Synonym Symbols: | CAR; CAR1; MB67 |
| NCBI Protein Information: | nuclear receptor subfamily 1 group I member 3; constitutive active receptor; constitutive active response; orphan nuclear receptor MB67; orphan nuclear hormone receptor; constitutive androstane receptor; constitutive activator of retinoid response; constitutive androstane nuclear receptor variant 2; constitutive androstane nuclear receptor variant 3; constitutive androstane nuclear receptor variant 4; constitutive androstane nuclear receptor variant 5 |
| UniProt Protein Name: | Nuclear receptor subfamily 1 group I member 3 |
| UniProt Synonym Protein Names: | Constitutive activator of retinoid response; Constitutive active response; Constitutive androstane receptor; CAR; Orphan nuclear receptor MB67 |
| Protein Family: | Nuclear receptor subfamily |
| UniProt Gene Name: | NR1I3 |
| UniProt Entry Name: | NR1I3_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 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.
| Step | Procedure |
| 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-NR1I3 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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