Cell Cycle ELISA Kits
MAT1 Colorimetric Cell-Based ELISA
- SKU:
- CBCAB01028
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Cell Cycle
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | MAT1 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01028 |
| ELISA Type: | Cell-Based |
| Target: | MAT1 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The MAT1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MAT1 protein expression profile in cells. The kit can be used for measuring the relative amounts of MAT1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MAT1.
Qualitative determination of MAT1 concentration is achieved by an indirect ELISA format. In essence, MAT1 is captured by MAT1-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: 4331, UniProt ID: P51948, OMIM: 602659, Unigene: Hs.509523 |
| Gene Symbol: | MNAT1 |
| Sub Type: | None |
| UniProt Protein Function: | MNAT1: Stabilizes the cyclin H-CDK7 complex to form a functional CDK-activating kinase (CAK) enzymatic complex. CAK activates the cyclin-associated kinases CDK1, CDK2, CDK4 and CDK6 by threonine phosphorylation. CAK complexed to the core-TFIIH basal transcription factor activates RNA polymerase II by serine phosphorylation of the repetitive C-terminus domain (CTD) of its large subunit (POLR2A), allowing its escape from the promoter and elongation of the transcripts. Involved in cell cycle control and in RNA transcription by RNA polymerase II. |
| UniProt Protein Details: | Protein type:Nuclear receptor co-regulator; Cell cycle regulation; Ubiquitin conjugating system; Ubiquitin ligase Chromosomal Location of Human Ortholog: 14q23 Cellular Component: nucleoplasm; holo TFIIH complex; cytoplasm Molecular Function:RNA polymerase subunit kinase activity; DNA-dependent ATPase activity; protein binding; zinc ion binding; protein N-terminus binding Biological Process: transcription from RNA polymerase II promoter; viral reproduction; positive regulation of viral transcription; positive regulation of smooth muscle cell proliferation; protein amino acid phosphorylation; regulation of gene expression, epigenetic; mRNA capping; negative regulation of gene expression, epigenetic; transcription-coupled nucleotide-excision repair; nucleotide-excision repair, DNA damage removal; protein complex assembly; G2/M transition of mitotic cell cycle; adult heart development; transcription initiation from RNA polymerase II promoter; ventricular system development; transcription from RNA polymerase I promoter; RNA elongation from RNA polymerase I promoter; termination of RNA polymerase I transcription; DNA repair; regulation of transcription from RNA polymerase II promoter; cell proliferation; nucleotide-excision repair; RNA elongation from RNA polymerase II promoter; gene expression; positive regulation of transcription from RNA polymerase II promoter; mitotic cell cycle; regulation of cyclin-dependent protein kinase activity; transcription initiation from RNA polymerase I promoter; response to calcium ion; negative regulation of apoptosis; G1/S transition of mitotic cell cycle |
| NCBI Summary: | The protein encoded by this gene, along with cyclin H and CDK7, forms the CDK-activating kinase (CAK) enzymatic complex. This complex activates several cyclin-associated kinases and can also associate with TFIIH to activate transcription by RNA polymerase II. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011] |
| UniProt Code: | P51948 |
| NCBI GenInfo Identifier: | 1708932 |
| NCBI Gene ID: | 4331 |
| NCBI Accession: | P51948.1 |
| UniProt Secondary Accession: | P51948,Q15817, Q6ICQ7, G3V1U8, |
| UniProt Related Accession: | P51948 |
| Molecular Weight: | 309 |
| NCBI Full Name: | CDK-activating kinase assembly factor MAT1 |
| NCBI Synonym Full Names: | MNAT CDK-activating kinase assembly factor 1 |
| NCBI Official Symbol: | MNAT1 |
| NCBI Official Synonym Symbols: | MAT1; TFB3; CAP35; RNF66 |
| NCBI Protein Information: | CDK-activating kinase assembly factor MAT1; RING finger protein 66; RING finger protein MAT1; CDK7/cyclin-H assembly factor; cyclin G1 interacting protein; cyclin-G1-interacting protein; menage a trois 1 (CAK assembly factor); menage a trois homolog 1, cyclin H assembly factor |
| UniProt Protein Name: | CDK-activating kinase assembly factor MAT1 |
| UniProt Synonym Protein Names: | CDK7/cyclin-H assembly factor; Cyclin-G1-interacting protein; Menage a trois; RING finger protein 66; RING finger protein MAT1; p35; p36 |
| Protein Family: | CDK-activating kinase assembly factor |
| UniProt Gene Name: | MNAT1 |
| UniProt Entry Name: | MAT1_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-MAT1 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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