Description
CDK10 Colorimetric Cell-Based ELISA
The CDK10 Colorimetric Cell-Based ELISA Kit from Assay Genie is a cutting-edge assay designed for the accurate detection of CDK10 levels in cell lysates and culture supernatants. This kit boasts high sensitivity and specificity, allowing for precise and reliable results that are essential for a variety of research applications.CDK10, also known as cyclin-dependent kinase 10, plays a crucial role in cell cycle regulation and cell growth. Dysregulation of CDK10 has been linked to various diseases, including cancer, making it a valuable biomarker for understanding disease progression and potential therapeutic targets.
With the CDK10 Colorimetric Cell-Based ELISA Kit, researchers can confidently measure CDK10 levels in their samples, providing valuable insights into cellular pathways and potential treatment strategies. Trust Assay Genie for accurate and reliable results in your CDK10 research.
| Product Name: | CDK10 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01105 |
| ELISA Type: | Cell-Based |
| Target: | CDK10 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The CDK10 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CDK10 protein expression profile in cells. The kit can be used for measuring the relative amounts of CDK10 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on CDK10.
Qualitative determination of CDK10 concentration is achieved by an indirect ELISA format. In essence, CDK10 is captured by CDK10-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: 8558, UniProt ID: Q15131, OMIM: 603464, Unigene: Hs.699177 |
| Gene Symbol: | CDK10 |
| Sub Type: | None |
| UniProt Protein Function: | CDK10: a CMGC kinase of the CDK family. Plays a role in cellular proliferation. Its function is limited to cell cycle G2-M phase. At least three alternatively spliced isoforms have been reported, two of which contain multiple non-AUG translation initiation sites. |
| UniProt Protein Details: | Protein type:Protein kinase, Ser/Thr (non-receptor); Protein kinase, CMGC; Cell cycle regulation; Kinase, protein; EC 2.7.11.22; CMGC group; CDK family; CDK/CDK10 subfamily; CDK10 subfamily Chromosomal Location of Human Ortholog: 16q24 Molecular Function:protein binding; cyclin-dependent protein kinase activity; ATP binding Biological Process: negative regulation of cell proliferation; traversing start control point of mitotic cell cycle; positive regulation of MAPKKK cascade; protein amino acid phosphorylation |
| NCBI Summary: | The protein encoded by this gene belongs to the CDK subfamily of the Ser/Thr protein kinase family. The CDK subfamily members are highly similar to the gene products of S. cerevisiae cdc28, and S. pombe cdc2, and are known to be essential for cell cycle progression. This kinase has been shown to play a role in cellular proliferation and its function is limited to cell cycle G2-M phase. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2009] |
| UniProt Code: | Q15131 |
| NCBI GenInfo Identifier: | 6226784 |
| NCBI Gene ID: | 8558 |
| NCBI Accession: | Q15131.1 |
| UniProt Secondary Accession: | Q15131,Q0VGZ7, Q15130, Q6PJC0, A8K370, A8K8I6, A8MXU6 B3KQJ3, B7Z420, D3DX82, D3DX83, |
| UniProt Related Accession: | Q15131 |
| Molecular Weight: | 272 |
| NCBI Full Name: | Cyclin-dependent kinase 10 |
| NCBI Synonym Full Names: | cyclin-dependent kinase 10 |
| NCBI Official Symbol: | CDK10 |
| NCBI Official Synonym Symbols: | PISSLRE |
| NCBI Protein Information: | cyclin-dependent kinase 10; CDC2-related protein kinase; cell division protein kinase 10; cyclin-dependent kinase (CDC2-like) 10; cyclin-dependent kinase related protein; serine/threonine protein kinase PISSLRE; serine/threonine-protein kinase PISSLRE |
| UniProt Protein Name: | Cyclin-dependent kinase 10 |
| UniProt Synonym Protein Names: | Cell division protein kinase 10; Serine/threonine-protein kinase PISSLRE |
| Protein Family: | Cyclin-dependent kinase |
| UniProt Gene Name: | CDK10 |
| UniProt Entry Name: | CDK10_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-CDK10 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.)
