Description
p50 Dynamitin Colorimetric Cell-Based ELISA Kit
The p50 Dynamitin Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers studying cellular dynamics and protein interactions. This kit allows for the detection of p50 Dynamitin in cell lysates and culture supernatants with high sensitivity and accuracy. By measuring the levels of p50 Dynamitin, researchers can gain insights into intracellular trafficking, vesicle transport, and cellular organization.P50 Dynamitin is a subunit of the dynactin complex, essential for dynein motor function and microtubule organization. Dysregulation of p50 Dynamitin has been implicated in various diseases, including neurodegenerative disorders and cancer.
Understanding its role in cellular processes can provide valuable information for disease mechanisms and potential therapeutic targets.The p50 Dynamitin Colorimetric Cell-Based ELISA Kit is easy to use, with clear instructions and fast results, making it a valuable tool for academic and pharmaceutical research. With its high specificity and reproducibility, this kit is a reliable option for studying p50 Dynamitin in a variety of biological samples.
| Product Name: | p50 Dynamitin Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00791 |
| ELISA Type: | Cell-Based |
| Target: | p50 Dynamitin |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The p50 Dynamitin Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect p50 Dynamitin protein expression profile in cells. The kit can be used for measuring the relative amounts of p50 Dynamitin in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on p50 Dynamitin.
Qualitative determination of p50 Dynamitin concentration is achieved by an indirect ELISA format. In essence, p50 Dynamitin is captured by p50 Dynamitin-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: 10540, UniProt ID: Q13561, OMIM: 138972, Unigene: Hs.429666/Hs.720728 |
| Gene Symbol: | DCTN2 |
| Sub Type: | None |
| UniProt Protein Function: | dynactin 2: Modulates cytoplasmic dynein binding to an organelle, and plays a role in prometaphase chromosome alignment and spindle organization during mitosis. Involved in anchoring microtubules to centrosomes. May play a role in synapse formation during brain development. Belongs to the dynactin subunit 2 family. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Cell cycle regulation; Motor; Microtubule-binding; Cytoskeletal Chromosomal Location of Human Ortholog: 12q13.3 Cellular Component: kinetochore; dynein complex; centrosome; microtubule; dynactin complex; growth cone; membrane; cytoplasm; cytosol; vesicle Molecular Function:protein binding; spectrin binding; motor activity Biological Process: cell proliferation; mitosis; mitotic spindle organization and biogenesis; metabolic process; organelle organization and biogenesis; antigen processing and presentation of exogenous peptide antigen via MHC class II; mitotic cell cycle; G2/M transition of mitotic cell cycle; melanosome transport |
| NCBI Summary: | This gene encodes a 50-kD subunit of dynactin, a macromolecular complex consisting of 10-11 subunits ranging in size from 22 to 150 kD. Dynactin binds to both microtubules and cytoplasmic dynein. It is involved in a diverse array of cellular functions, including ER-to-Golgi transport, the centripetal movement of lysosomes and endosomes, spindle formation, chromosome movement, nuclear positioning, and axonogenesis. This subunit is present in 4-5 copies per dynactin molecule. It contains three short alpha-helical coiled-coil domains that may mediate association with self or other dynactin subunits. It may interact directly with the largest subunit (p150) of dynactin and may affix p150 in place. Multiple alternatively spliced transcript variants encoding distinct isoforms have been found for this gene. [provided by RefSeq, May 2012] |
| UniProt Code: | Q13561 |
| NCBI GenInfo Identifier: | 22096346 |
| NCBI Gene ID: | 10540 |
| NCBI Accession: | Q13561.4 |
| UniProt Secondary Accession: | Q13561,Q86YN2, Q9BW17, B2RBK5, |
| UniProt Related Accession: | Q13561 |
| Molecular Weight: | 401 |
| NCBI Full Name: | Dynactin subunit 2 |
| NCBI Synonym Full Names: | dynactin 2 (p50) |
| NCBI Official Symbol: | DCTN2 |
| NCBI Official Synonym Symbols: | RBP50; DCTN50; HEL-S-77; DYNAMITIN |
| NCBI Protein Information: | dynactin subunit 2; p50 dynamitin; dynactin complex 50 kD subunit; dynactin complex 50 kDa subunit; epididymis secretory protein Li 77; 50 kD dynein-associated polypeptide; 50 kDa dynein-associated polypeptide |
| UniProt Protein Name: | Dynactin subunit 2 |
| UniProt Synonym Protein Names: | 50 kDa dynein-associated polypeptide; Dynactin complex 50 kDa subunit; DCTN-50; p50 dynamitin |
| Protein Family: | Dynactin |
| UniProt Gene Name: | DCTN2 |
| UniProt Entry Name: | DCTN2_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-p50 Dynamitin 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.)
