Description
KIF20A Colorimetric Cell-Based ELISA Kit
The KIF20A Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the quantitative detection of KIF20A levels in cell lysates and culture supernatants. This kit offers exceptional accuracy and precision, allowing researchers to confidently analyze the expression of KIF20A in various cell types and conditions.KIF20A, or kinesin family member 20A, is a motor protein involved in cell division and cytokinesis. Its dysregulation has been implicated in cancer progression and other diseases, making it a valuable target for research and drug development.
The KIF20A Colorimetric Cell-Based ELISA Kit provides a powerful tool for studying the role of KIF20A in cellular processes and disease mechanisms.With its user-friendly protocol and robust performance, the KIF20A Colorimetric Cell-Based ELISA Kit is an essential tool for researchers investigating the functions of KIF20A and its potential implications in disease. Elevate your research with this advanced assay kit from AssayGenie.
| Product Name: | KIF20A Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00731 |
| ELISA Type: | Cell-Based |
| Target: | KIF20A |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The KIF20A Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect KIF20A protein expression profile in cells. The kit can be used for measuring the relative amounts of KIF20A in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on KIF20A.
Qualitative determination of KIF20A concentration is achieved by an indirect ELISA format. In essence, KIF20A is captured by KIF20A-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: 10112, UniProt ID: O95235, OMIM: 605664, Unigene: Hs.718626 |
| Gene Symbol: | KIF20A |
| Sub Type: | None |
| UniProt Protein Function: | KIF20A: Mitotic kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis. Following phosphorylation by PLK1, involved in recruitment of PLK1 to the central spindle. Interacts with guanosine triphosphate (GTP)-bound forms of RAB6A and RAB6B. May act as a motor required for the retrograde RAB6 regulated transport of Golgi membranes and associated vesicles along microtubules. Has a microtubule plus end-directed motility. Belongs to the kinesin-like protein family. |
| UniProt Protein Details: | Protein type:Motor; Microtubule-binding Chromosomal Location of Human Ortholog: 5q31 Cellular Component: kinesin complex; midbody; nucleoplasm; spindle Molecular Function:ATPase activity; microtubule motor activity; protein binding; protein kinase binding; transporter activity Biological Process: cell separation during cytokinesis; cytokinesis; cytokinesis after mitosis; microtubule bundle formation; microtubule-based movement; vesicle-mediated transport |
| UniProt Code: | O95235 |
| NCBI GenInfo Identifier: | 6225915 |
| NCBI Gene ID: | 10112 |
| NCBI Accession: | O95235.1 |
| UniProt Secondary Accession: | O95235,B4DL79, D3DQB6, |
| UniProt Related Accession: | O95235 |
| Molecular Weight: | 98,100 Da |
| NCBI Full Name: | Kinesin-like protein KIF20A |
| NCBI Synonym Full Names: | kinesin family member 20A |
| NCBI Official Symbol: | KIF20A |
| NCBI Official Synonym Symbols: | MKLP2; RAB6KIFL |
| NCBI Protein Information: | kinesin-like protein KIF20A |
| UniProt Protein Name: | Kinesin-like protein KIF20A |
| UniProt Synonym Protein Names: | GG10_2; Mitotic kinesin-like protein 2; MKlp2; Rab6-interacting kinesin-like protein; Rabkinesin-6 |
| Protein Family: | Kinesin-like protein |
| UniProt Gene Name: | KIF20A |
| UniProt Entry Name: | KI20A_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-KIF20A 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.)
