Description
DOK7 Colorimetric Cell-Based ELISA
The DOK7 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the quantitative detection of DOK7 protein levels in cell lysates. This kit offers high sensitivity and specificity, providing accurate and reproducible results for researchers studying cellular signaling pathways and protein interactions.DOK7 is a key regulator of neuromuscular synapse development and maintenance, playing a critical role in skeletal muscle function.
Dysregulation of DOK7 has been linked to various neuromuscular disorders, making it a valuable target for therapeutic interventions and drug development.With easy-to-follow protocols and user-friendly instructions, the DOK7 Colorimetric Cell-Based ELISA Kit is suitable for a wide range of cell biology research applications. Trust in AssayGenie's expertise and innovation to accelerate your research and advance scientific discoveries.
| Product Name: | DOK7 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01124 |
| ELISA Type: | Cell-Based |
| Target: | DOK7 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The DOK7 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect DOK7 protein expression profile in cells. The kit can be used for measuring the relative amounts of DOK7 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on DOK7.
Qualitative determination of DOK7 concentration is achieved by an indirect ELISA format. In essence, DOK7 is captured by DOK7-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: 285489, UniProt ID: Q18PE1, OMIM: 254300/610285, Unigene: Hs.122110/Hs.701584 |
| Gene Symbol: | DOK7 |
| Sub Type: | None |
| UniProt Protein Function: | DOK7: docking proteins are enzymatically inert adaptor or scaffolding proteins. They provide a docking platform for the assembly of multimolecular signaling complexes. Probable muscle-intrinsic activator of MUSK that plays an essential role in neuromuscular synaptogenesis. Acts in aneural activation of MUSK and subsequent acetylcholine receptor (AchR) clustering in myotubes. Induces autophosphorylation of MUSK. Interacts with the cytoplasmic part of MUSK. Defects in DOK7 are the cause of congenital myasthenic syndrome type 1B or CMS1B. Three isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Adaptor/scaffold; Activator Chromosomal Location of Human Ortholog: 4p16.3 Cellular Component: cell junction; plasma membrane; synapse Molecular Function:insulin receptor binding; lipid binding; protein kinase binding Disease: Fetal Akinesia Deformation Sequence; Myasthenia, Limb-girdle, Familial |
| NCBI Summary: | The protein encoded by this gene is essential for neuromuscular synaptogenesis. The protein functions in aneural activation of muscle-specific receptor kinase, which is required for postsynaptic differentiation, and in the subsequent clustering of the acetylcholine receptor in myotubes. This protein can also induce autophosphorylation of muscle-specific receptor kinase. Mutations in this gene are a cause of familial limb-girdle myasthenia autosomal recessive, which is also known as congenital myasthenic syndrome type 1B. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Sep 2009] |
| UniProt Code: | Q18PE1 |
| NCBI GenInfo Identifier: | 115311705 |
| NCBI Gene ID: | 285489 |
| NCBI Accession: | Q18PE1.1 |
| UniProt Secondary Accession: | Q18PE1,Q6P6A6, Q86XG5, Q8N2J3, Q8NBC1, A2A499, A2RRD4 E9PB56, |
| UniProt Related Accession: | Q18PE1 |
| Molecular Weight: | 63,938 Da |
| NCBI Full Name: | Protein Dok-7 |
| NCBI Synonym Full Names: | docking protein 7 |
| NCBI Official Symbol: | DOK7 |
| NCBI Official Synonym Symbols: | CMS10; CMS1B; C4orf25 |
| NCBI Protein Information: | protein Dok-7 |
| UniProt Protein Name: | Protein Dok-7 |
| UniProt Synonym Protein Names: | Downstream of tyrosine kinase 7 |
| Protein Family: | Protein |
| UniProt Gene Name: | DOK7 |
| UniProt Entry Name: | DOK7_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-DOK7 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.)
