Description
Nuclear Receptor NR4A1 Colorimetric Cell-Based ELISA Kit
The Nuclear Receptor NR4A1 Colorimetric Cell-Based ELISA Kit is a powerful tool for studying the expression levels of NR4A1 in cell samples. This kit provides precise and reliable measurements of NR4A1 levels in a colorimetric format, allowing researchers to accurately assess the activity of this nuclear receptor.NR4A1, also known as Nur77, is a transcription factor that plays a key role in various biological processes, including cell proliferation, apoptosis, and inflammation. Dysregulation of NR4A1 has been linked to several diseases, including cancer, metabolic disorders, and inflammatory conditions.
By using this ELISA kit, researchers can gain valuable insights into the role of NR4A1 in these diseases and potentially uncover new therapeutic targets.With its high sensitivity and specificity, the Nuclear Receptor NR4A1 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers studying the function and regulation of NR4A1 in different cellular contexts. Whether investigating the molecular mechanisms of NR4A1 or screening for potential therapeutic compounds, this kit offers accurate and reproducible results to advance your research goals.
| Product Name: | Nuclear Receptor NR4A1 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00186 |
| ELISA Type: | Cell-Based |
| Target: | Nuclear Receptor NR4A1 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Nuclear Receptor NR4A1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Nuclear Receptor NR4A1 protein expression profile in cells. The kit can be used for measuring the relative amounts of Nuclear Receptor NR4A1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Nuclear Receptor NR4A1.
Qualitative determination of Nuclear Receptor NR4A1 concentration is achieved by an indirect ELISA format. In essence, Nuclear Receptor NR4A1 is captured by Nuclear Receptor NR4A1-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: 3164, UniProt ID: P22736, OMIM: 139139, Unigene: Hs.524430 |
| Gene Symbol: | NR4A1 |
| Sub Type: | None |
| UniProt Protein Function: | Nur77: an orphan nuclear receptor and immediate-early gene that regulates cellular proliferation, apoptosis, inflammation, and glucose metabolism. Induced by exercise in muscle and is a functional regulator of glucose metabolism in skeletal muscle. Its level decreases in the muscle of obese insulin-resistant men. Acts concomitantly with NURR1 in regulating the expression of delayed-early genes during liver regeneration. Binds the NGFI-B response element (NBRE) 5'-AAAAGGTCA-3'. May inhibit NF-kappa-B transactivation of IL2. A mediator of TCR-directed thymocyte apoptosis. TCR-signaling induces a FAIM/Akt/Nur77 signaling pathway that is critical for modulating apoptosis in developing thymocytes. A physiological substrate of the MEK-ERK-RSK cascade that modulates nuclear export and intracellular translocation during T cell death. Binds DNA as a monomer. Interacts with GADD45GIP1. Overexpression of Nur77 induces the expression of both p300 and HDAC1. Acetylation by p300 and HDAC1 may regulate the rapid turnover of Nur77 protein. |
| UniProt Protein Details: | Protein type:Nuclear receptor; Apoptosis; DNA-binding Chromosomal Location of Human Ortholog: 12q13 Cellular Component: nucleoplasm; nuclear membrane; cytoplasm; nucleus Molecular Function:protein binding; ligand-dependent nuclear receptor activity; DNA binding; zinc ion binding; sequence-specific DNA binding; steroid hormone receptor activity Biological Process: epidermal growth factor receptor signaling pathway; transcription initiation from RNA polymerase II promoter; phosphoinositide-mediated signaling; intracellular receptor-mediated signaling pathway; fibroblast growth factor receptor signaling pathway; cell migration during sprouting angiogenesis; nerve growth factor receptor signaling pathway; innate immune response; positive regulation of endothelial cell proliferation; positive regulation of transcription from RNA polymerase II promoter; steroid hormone mediated signaling; gene expression; signal transduction |
| NCBI Summary: | This gene encodes a member of the steroid-thyroid hormone-retinoid receptor superfamily. Expression is induced by phytohemagglutinin in human lymphocytes and by serum stimulation of arrested fibroblasts. The encoded protein acts as a nuclear transcription factor. Translocation of the protein from the nucleus to mitochondria induces apoptosis. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2011] |
| UniProt Code: | P22736 |
| NCBI GenInfo Identifier: | 127819 |
| NCBI Gene ID: | 3164 |
| NCBI Accession: | P22736.1 |
| UniProt Secondary Accession: | P22736,Q15627, B4DML7, |
| UniProt Related Accession: | P22736 |
| Molecular Weight: | 64,463 Da |
| NCBI Full Name: | Nuclear receptor subfamily 4 group A member 1 |
| NCBI Synonym Full Names: | nuclear receptor subfamily 4, group A, member 1 |
| NCBI Official Symbol: | NR4A1 |
| NCBI Official Synonym Symbols: | HMR; N10; TR3; NP10; GFRP1; NAK-1; NGFIB; NUR77 |
| NCBI Protein Information: | nuclear receptor subfamily 4 group A member 1; ST-59; hormone receptor; TR3 orphan receptor; steroid receptor TR3; testicular receptor 3; early response protein NAK1; orphan nuclear receptor HMR; orphan nuclear receptor TR3; nuclear hormone receptor NUR/77; growth factor-inducible nuclear protein N10; nerve growth factor IB nuclear receptor variant 1 |
| UniProt Protein Name: | Nuclear receptor subfamily 4 group A member 1 |
| UniProt Synonym Protein Names: | Early response protein NAK1; Nuclear hormone receptor NUR/77; Nur77; Orphan nuclear receptor HMR; Orphan nuclear receptor TR3; ST-59; Testicular receptor 3 |
| Protein Family: | Nuclear receptor subfamily |
| UniProt Gene Name: | NR4A1 |
| UniProt Entry Name: | NR4A1_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-Nuclear Receptor NR4A1 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.)
