Description
CNOT2 Colorimetric Cell-Based ELISA Kit
The CNOT2 Colorimetric Cell Based ELISA Kit is a highly sensitive and specific assay designed for the accurate detection of CNOT2 levels in cell lysates and culture supernatants. This kit offers reliable and reproducible results, making it an ideal tool for a variety of research applications.CNOT2 is a key component of the CCR4-NOT complex involved in mRNA degradation and regulation of gene expression. Dysregulation of CNOT2 has been implicated in various diseases, including cancer and neurological disorders, making it a valuable biomarker for studying these conditions and potential therapeutic interventions.
With its ease of use and high performance, the CNOT2 Colorimetric Cell Based ELISA Kit is a valuable tool for researchers interested in unraveling the role of CNOT2 in disease pathogenesis and exploring new treatment avenues.
| Product Name: | CNOT2 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00590 |
| ELISA Type: | Cell-Based |
| Target: | CNOT2 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The CNOT2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CNOT2 protein expression profile in cells. The kit can be used for measuring the relative amounts of CNOT2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on CNOT2.
Qualitative determination of CNOT2 concentration is achieved by an indirect ELISA format. In essence, CNOT2 is captured by CNOT2-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: 4848, UniProt ID: Q9NZN8, OMIM: 604909, Unigene: Hs.133350 |
| Gene Symbol: | CNOT2 |
| Sub Type: | None |
| UniProt Protein Function: | NOT2: The CCR4-NOT complex functions as general transcription regulation complex. Subunit of the CCR4-NOT core complex that contains CHAF1A, CHAF1B, CNOT1, CNOT2, CNOT3, CNOT4, CNOT6 and CNOT8. Ubiquitous. Highly expressed in brain, heart, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocytes. Belongs to the CNOT2/3/5 family. 5 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Transcription, coactivator/corepressor Chromosomal Location of Human Ortholog: 12q15 Cellular Component: CCR4-NOT complex; cytoplasm; cytosol; membrane; nucleus; plasma membrane Molecular Function:poly(A)-specific ribonuclease activity; protein binding Biological Process: DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; mRNA catabolic process, deadenylation-dependent decay; negative regulation of estrogen receptor signaling pathway; negative regulation of transcription from RNA polymerase II promoter; negative regulation of translation; poly(A) tail shortening |
| NCBI Summary: | This gene encodes a subunit of the multi-component CCR4-NOT complex. The CCR4-NOT complex regulates mRNA synthesis and degradation and is also thought to be involved in mRNA splicing, transport and localization. The encoded protein interacts with histone deacetylases and functions as a repressor of polymerase II transcription. Alternatively spliced transcript variants have been observed for this gene. [provided by RefSeq, Dec 2010] |
| UniProt Code: | Q9NZN8 |
| NCBI GenInfo Identifier: | 46396017 |
| NCBI Gene ID: | 4848 |
| NCBI Accession: | Q9NZN8.1 |
| UniProt Secondary Accession: | Q9NZN8,Q9H3E0, Q9NSX5, Q9NWR6, Q9P028, |
| UniProt Related Accession: | Q9NZN8 |
| Molecular Weight: | 60kDa |
| NCBI Full Name: | CCR4-NOT transcription complex subunit 2 |
| NCBI Synonym Full Names: | CCR4-NOT transcription complex subunit 2 |
| NCBI Official Symbol: | CNOT2Â Â |
| NCBI Official Synonym Symbols: | NOT2; CDC36; NOT2H; HSPC131Â Â |
| NCBI Protein Information: | CCR4-NOT transcription complex subunit 2 |
| UniProt Protein Name: | CCR4-NOT transcription complex subunit 2 |
| UniProt Synonym Protein Names: | CCR4-associated factor 2 |
| Protein Family: | CCR4-NOT transcription complex |
| UniProt Gene Name: | CNOT2Â Â |
| 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-CNOT2 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.)
