Cell Cycle ELISA Kits
Cyclin E2 Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB00612
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Cell Cycle
- Reactivity:
- Human
- Reactivity:
- Mouse
- Detection Method:
- Colorimetric
Description
| Product Name: | Cyclin E2 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00612 |
| ELISA Type: | Cell-Based |
| Target: | Cyclin E2 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Cyclin E2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cyclin E2 protein expression profile in cells. The kit can be used for measuring the relative amounts of Cyclin E2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cyclin E2.
Qualitative determination of Cyclin E2 concentration is achieved by an indirect ELISA format. In essence, Cyclin E2 is captured by Cyclin E2-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: 9134, UniProt ID: O96020, OMIM: 603775, Unigene: Hs.567387 |
| Gene Symbol: | CCNE2 |
| Sub Type: | None |
| UniProt Protein Function: | CCNE2: Essential for the control of the cell cycle at the late G1 and early S phase. Interacts with the CDK2 (in vivo) and CDK3 (in vitro) protein kinases to form a serine/threonine kinase holoenzyme complex. The cyclin subunit imparts substrate specificity to the complex. Activated by papilloma viral oncoproteins E6 and E7 which bind to and inactivate p53 and Rb, respectively. According to PubMed:9858585, highest levels of expression in adult testis, thymus and brain. Lower levels in placenta, spleen and colon. Consistently elevated levels in tumor- derived cells compared to non-transformed proliferating cells. According to PubMed:9840927: low levels in thymus, prostate, brain, skeletal muscle, and kidney. Elevated levels in lung. According to PubMed:9840943 highly expressed in testis, placenta, thymus and brain. In a lesser extent in small intestine and colon. Belongs to the cyclin family. Cyclin E subfamily. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Cell cycle regulation; Activator Chromosomal Location of Human Ortholog: 8q22.1 Cellular Component: nucleoplasm; cytosol Molecular Function:protein binding; cyclin-dependent protein kinase regulator activity Biological Process: DNA replication initiation; cell division; regulation of cyclin-dependent protein kinase activity; mitotic cell cycle; cell cycle checkpoint; G1/S transition of mitotic cell cycle |
| NCBI Summary: | The protein encoded by this gene belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. This cyclin forms a complex with and functions as a regulatory subunit of CDK2. This cyclin has been shown to specifically interact with CIP/KIP family of CDK inhibitors, and plays a role in cell cycle G1/S transition. The expression of this gene peaks at the G1-S phase and exhibits a pattern of tissue specificity distinct from that of cyclin E1. A significantly increased expression level of this gene was observed in tumor-derived cells. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O96020 |
| NCBI GenInfo Identifier: | 5921733 |
| NCBI Gene ID: | 9134 |
| NCBI Accession: | O96020.1 |
| UniProt Secondary Accession: | O96020,O95439, |
| UniProt Related Accession: | O96020 |
| Molecular Weight: | 404 |
| NCBI Full Name: | G1/S-specific cyclin-E2 |
| NCBI Synonym Full Names: | cyclin E2 |
| NCBI Official Symbol: | CCNE2 |
| NCBI Official Synonym Symbols: | CYCE2 |
| NCBI Protein Information: | G1/S-specific cyclin-E2 |
| UniProt Protein Name: | G1/S-specific cyclin-E2 |
| Protein Family: | G1/S-specific cyclin |
| UniProt Gene Name: | CCNE2 |
| UniProt Entry Name: | CCNE2_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-Cyclin E2 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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