Description
NR2F6 Colorimetric Cell-Based ELISA
The NR2F6 Colorimetric Cell-Based ELISA Kit is a powerful tool for detecting and quantifying NR2F6 protein levels in cell lysates and tissue samples. This kit offers high sensitivity and specificity, providing accurate and reliable results for research applications.NR2F6, also known as COUP-TFII, is a transcription factor that plays a critical role in various biological processes, including embryonic development, metabolism, and immune responses.
Dysregulation of NR2F6 has been linked to various diseases, making it a valuable biomarker for studying these conditions and developing targeted therapies.With easy-to-follow protocols and quick assay times, the NR2F6 Colorimetric Cell-Based ELISA Kit is suitable for both experienced researchers and beginners in the field. Unlock the potential of your research by accurately measuring NR2F6 levels with this innovative ELISA kit.
| Product Name: | NR2F6 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00134 |
| ELISA Type: | Cell-Based |
| Target: | NR2F6 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The NR2F6 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NR2F6 protein expression profile in cells. The kit can be used for measuring the relative amounts of NR2F6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NR2F6.
Qualitative determination of NR2F6 concentration is achieved by an indirect ELISA format. In essence, NR2F6 is captured by NR2F6-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: 2063, UniProt ID: P10588, OMIM: 132880, Unigene: Hs.466148 |
| Gene Symbol: | NR2F6 |
| Sub Type: | None |
| UniProt Protein Function: | EAR-2: Transcription factor predominantly involved in transcriptional repression. Binds to promoter/enhancer response elements that contain the imperfect 5'-AGGTCA-3' direct or inverted repeats with various spacings which are also recognized by other nuclear hormone receptors. Involved in modulation of hormonal responses. Represses transcriptional activity of the lutropin-choriogonadotropic hormone receptor/LHCGR gene, the renin/REN gene and the oxytocin-neurophysin/OXT gene. Represses the triiodothyronine-dependent and -independent transcriptional activity of the thyroid hormone receptor gene in a cell type- specific manner. The corepressing function towards thyroid hormone receptor beta/THRB involves at least in part the inhibition of THRB binding to triiodothyronine response elements (TREs) by NR2F6. Inhibits NFATC transcription factor DNA binding and subsequently its transcriptional activity. Acts as transcriptional repressor of IL-17 expression in Th-17 differentiated CD4(+) T cells and may be involved in induction and/or maintenance of peripheral immunological tolerance and autoimmunity. Involved in development of forebrain circadian clock; is required early in the development of the locus coeruleus (LC). Belongs to the nuclear hormone receptor family. NR2 subfamily. |
| UniProt Protein Details: | Protein type:Nuclear receptor; DNA-binding Chromosomal Location of Human Ortholog: 19p13.1 Cellular Component: nucleoplasm; nucleus Molecular Function:DNA binding; ligand-dependent nuclear receptor activity; protein binding; sequence-specific DNA binding; thyroid hormone receptor activity; transcription factor activity Biological Process: negative regulation of transcription from RNA polymerase II promoter; signal transduction; transcription initiation from RNA polymerase II promoter |
| UniProt Code: | P10588 |
| NCBI GenInfo Identifier: | 23503053 |
| NCBI Gene ID: | 2063 |
| NCBI Accession: | P10588.2 |
| UniProt Secondary Accession: | P10588,Q5XGA0, Q6P586, Q9BUE8, B2RC68, |
| UniProt Related Accession: | P10588 |
| Molecular Weight: | 42,979 Da |
| NCBI Full Name: | Nuclear receptor subfamily 2 group F member 6 |
| NCBI Synonym Full Names: | nuclear receptor subfamily 2 group F member 6 |
| NCBI Official Symbol: | NR2F6 |
| NCBI Official Synonym Symbols: | EAR2; EAR-2; ERBAL2 |
| NCBI Protein Information: | nuclear receptor subfamily 2 group F member 6 |
| UniProt Protein Name: | Nuclear receptor subfamily 2 group F member 6 |
| UniProt Synonym Protein Names: | V-erbA-related protein 2; EAR-2 |
| Protein Family: | Nuclear receptor subfamily |
| UniProt Gene Name: | NR2F6 |
| UniProt Entry Name: | NR2F6_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-NR2F6 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.)
