Description
Cyclin G Colorimetric Cell-Based ELISA Kit
The Cyclin G Colorimetric Cell-Based ELISA Kit is a powerful tool for the detection of cyclin G levels in cell lysates and culture supernatants. This kit offers exceptional sensitivity and specificity, providing researchers with accurate and reliable results for a variety of experimental applications.Cyclin G is a key regulator of the cell cycle and has been implicated in various cellular processes, including cell proliferation, apoptosis, and DNA damage response.
Dysregulation of cyclin G expression has been associated with numerous diseases, including cancer and neurological disorders, making it a valuable target for therapeutic interventions.By utilizing the Cyclin G Colorimetric Cell-Based ELISA Kit, researchers can effectively study the role of cyclin G in cellular processes and disease pathogenesis, ultimately advancing our understanding of these conditions and potentially identifying new treatment strategies.
| Product Name: | Cyclin G Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00613 |
| ELISA Type: | Cell-Based |
| Target: | Cyclin G |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Cyclin G Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cyclin G protein expression profile in cells. The kit can be used for measuring the relative amounts of Cyclin G in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cyclin G.
Qualitative determination of Cyclin G concentration is achieved by an indirect ELISA format. In essence, Cyclin G is captured by Cyclin G-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: 900, UniProt ID: P51959, OMIM: 601578, Unigene: Hs.79101 |
| Gene Symbol: | CCNG1 |
| Sub Type: | None |
| UniProt Protein Function: | CCNG1: May play a role in growth regulation. Is associated with G2/M phase arrest in response to DNA damage. May be an intermediate by which p53 mediates its role as an inhibitor of cellular proliferation. Belongs to the cyclin family. Cyclin G subfamily. |
| UniProt Protein Details: | Protein type:Cell cycle regulation Chromosomal Location of Human Ortholog: 5q32-q34 Cellular Component: nucleoplasm Molecular Function:protein binding Biological Process: regulation of cyclin-dependent protein kinase activity |
| NCBI Summary: | The eukaryotic cell cycle is governed by cyclin-dependent protein kinases (CDKs) whose activities are regulated by cyclins and CDK inhibitors. The protein encoded by this gene is a member of the cyclin family and contains the cyclin box. The encoded protein lacks the protein destabilizing (PEST) sequence that is present in other family members. Transcriptional activation of this gene can be induced by tumor protein p53. Two transcript variants encoding the same protein have been identified for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P51959 |
| NCBI GenInfo Identifier: | 2506334 |
| NCBI Gene ID: | 900 |
| NCBI Accession: | P51959.2 |
| UniProt Secondary Accession: | P51959,Q15757, Q96L32, B2R7B2, B4DLW7, D3DQK7, |
| UniProt Related Accession: | P51959 |
| Molecular Weight: | 18,769 Da |
| NCBI Full Name: | Cyclin-G1 |
| NCBI Synonym Full Names: | cyclin G1 |
| NCBI Official Symbol: | CCNG1Â Â |
| NCBI Official Synonym Symbols: | CCNGÂ Â |
| NCBI Protein Information: | cyclin-G1 |
| UniProt Protein Name: | Cyclin-G1 |
| Protein Family: | Cyclin |
| UniProt Gene Name: | CCNG1Â Â |
| UniProt Entry Name: | CCNG1_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 G 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.)
