Developmental Biology
Doublecortin Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB00625
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Developmental Biology
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | Doublecortin Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00625 |
| ELISA Type: | Cell-Based |
| Target: | Doublecortin |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Doublecortin Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Doublecortin protein expression profile in cells. The kit can be used for measuring the relative amounts of Doublecortin in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Doublecortin.
Qualitative determination of Doublecortin concentration is achieved by an indirect ELISA format. In essence, Doublecortin is captured by Doublecortin-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: 1641, UniProt ID: O43602, OMIM: 300121, Unigene: Hs.34780 |
| Gene Symbol: | DCX |
| Sub Type: | None |
| UniProt Protein Function: | Doublecortin: Microtubule-associated protein required for initial steps of neuronal dispersion and cortex lamination during cerebral cortex development. May act by competing with the putative neuronal protein kinase DCAMKL1 in binding to a target protein. May in that way participate in a signaling pathway that is crucial for neuronal interaction before and during migration, possibly as part of a calcium ion-dependent signal transduction pathway. May be part with LIS-1 of a overlapping, but distinct, signaling pathways that promote neuronal migration. Interacts with tubulin. Highly expressed in neuronal cells of fetal brain (in the majority of cells of the cortical plate, intermediate zone and ventricular zone), but not expressed in other fetal tissues. In the adult, highly expressed in the brain frontal lobe, but very low expression in other regions of brain, and not detected in heart, placenta, lung, liver, skeletal muscles, kidney and pancreas. 5 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Cytoskeletal; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: Xq22.3-q23 Cellular Component: microtubule; microtubule associated complex; neuron projection; cytoskeleton; cytosol Molecular Function:protein binding; microtubule binding; protein kinase binding Biological Process: nervous system development; axon guidance; central nervous system development; axon extension; dendrite morphogenesis; neuron migration; central nervous system projection neuron axonogenesis; brain development Disease: Lissencephaly, X-linked, 1 |
| NCBI Summary: | This gene encodes a member of the doublecortin family. The protein encoded by this gene is a cytoplasmic protein and contains two doublecortin domains, which bind microtubules. In the developing cortex, cortical neurons must migrate over long distances to reach the site of their final differentiation. The encoded protein appears to direct neuronal migration by regulating the organization and stability of microtubules. In addition, the encoded protein interacts with LIS1, the regulatory gamma subunit of platelet activating factor acetylhydrolase, and this interaction is important to proper microtubule function in the developing cortex. Mutations in this gene cause abnormal migration of neurons during development and disrupt the layering of the cortex, leading to epilepsy, mental retardation, subcortical band heterotopia ("double cortex" syndrome) in females and lissencephaly ("smooth brain" syndrome) in males. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2010] |
| UniProt Code: | O43602 |
| NCBI GenInfo Identifier: | 215274172 |
| NCBI Gene ID: | 1641 |
| NCBI Accession: | O43602.3 |
| UniProt Secondary Accession: | O43602,O43911, Q5JYZ5, A6NFY6, A9Z1V8, D3DUY8, D3DUY9 D3DUZ0, |
| UniProt Related Accession: | O43602 |
| Molecular Weight: | 441 |
| NCBI Full Name: | Neuronal migration protein doublecortin |
| NCBI Synonym Full Names: | doublecortin |
| NCBI Official Symbol: | DCX |
| NCBI Official Synonym Symbols: | DC; DBCN; LISX; SCLH; XLIS |
| NCBI Protein Information: | neuronal migration protein doublecortin; lis-X; doublin; doublecortex; lissencephalin-X |
| UniProt Protein Name: | Neuronal migration protein doublecortin |
| UniProt Synonym Protein Names: | Doublin; Lissencephalin-X; Lis-X |
| Protein Family: | Protein doublecortin |
| UniProt Gene Name: | DCX |
| UniProt Entry Name: | DCX_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-Doublecortin 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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