Description
AAK1 Colorimetric Cell-Based ELISA
The AAK1 Colorimetric Cell-based ELISA Kit is a cutting-edge tool for researchers looking to accurately measure the levels of AAK1 (AP2 associated kinase 1) in cell culture supernatants. This kit is specifically designed for use with human samples, offering high sensitivity and specificity for reliable results.AAK1 is a key enzyme involved in clathrin-mediated endocytosis, playing a crucial role in various cellular processes. Dysregulation of AAK1 has been implicated in diseases such as cancer, neurological disorders, and viral infections, making it a valuable target for therapeutic development.
With the AAK1 Colorimetric Cell-based ELISA Kit, researchers can gain valuable insights into the role of AAK1 in disease pathology and explore its potential as a therapeutic target. This kit is easy to use, providing accurate and reproducible results for a wide range of research applications.
| Product Name: | AAK1 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01125 |
| ELISA Type: | Cell-Based |
| Target: | AAK1 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The AAK1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect AAK1 protein expression profile in cells. The kit can be used for measuring the relative amounts of AAK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on AAK1.
Qualitative determination of AAK1 concentration is achieved by an indirect ELISA format. In essence, AAK1 is captured by AAK1-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: 22848, UniProt ID: Q2M2I8, OMIM: 0, Unigene: Hs.468878 |
| Gene Symbol: | AAK1 |
| Sub Type: | None |
| UniProt Protein Function: | AAK1: an NAK kinase. Plays a regulatory role in clathrin-mediated endocytosis, including the regulation of fatty acid synthesis and cholesterol synthesis. |
| UniProt Protein Details: | Protein type:Kinase, protein; Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.1; Protein kinase, Other; Other group; NAK family Chromosomal Location of Human Ortholog: 2p14 Cellular Component: clathrin-coated vesicle; cytoplasm; extrinsic to plasma membrane; leading edge; terminal button Molecular Function:Notch binding; protein binding; protein serine/threonine kinase activity Biological Process: positive regulation of Notch signaling pathway; protein amino acid autophosphorylation; protein amino acid phosphorylation; protein stabilization; regulation of protein localization |
| NCBI Summary: | Adaptor-related protein complex 2 (AP-2 complexes) functions during receptor-mediated endocytosis to trigger clathrin assembly, interact with membrane-bound receptors, and recruit encodytic accessory factors. This gene encodes a member of the SNF1 subfamily of Ser/Thr protein kinases. The protein interacts with and phosphorylates a subunit of the AP-2 complex, which promotes binding of AP-2 to sorting signals found in membrane-bound receptors and subsequent receptor endocytosis. Its kinase activity is stimulated by clathrin. Alternatively spliced transcript variants have been described, but their biological validity has not been determined. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q2M2I8 |
| NCBI GenInfo Identifier: | 300669613 |
| NCBI Gene ID: | 22848 |
| NCBI Accession: | Q2M2I8.3 |
| UniProt Secondary Accession: | Q2M2I8,Q4ZFZ3, Q53RX6, Q9UPV4, |
| UniProt Related Accession: | Q2M2I8 |
| Molecular Weight: | 93,578 Da |
| NCBI Full Name: | AP2-associated protein kinase 1 |
| NCBI Synonym Full Names: | AP2 associated kinase 1 |
| NCBI Official Symbol: | AAK1 |
| NCBI Protein Information: | AP2-associated protein kinase 1 |
| UniProt Protein Name: | AP2-associated protein kinase 1 |
| UniProt Synonym Protein Names: | Adaptor-associated kinase 1 |
| Protein Family: | AP2-associated protein kinase |
| UniProt Gene Name: | AAK1 |
| UniProt Entry Name: | AAK1_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-AAK1 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.)
