Description
ACK1 (Phospho-Tyr284)Colorimetric Cell-Based ELISA Kit
The ACK1 Phospho-Tyr284 Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate detection of phosphorylated Ack1 (Activated Cdc42-associated tyrosine kinase 1) at tyrosine 284 levels in cell lysates and tissue homogenates. This kit offers high sensitivity and specificity, ensuring precise and reproducible results that are essential for various cell signaling studies.Ack1 is a key tyrosine kinase that plays a critical role in cell signaling pathways involved in cell growth, survival, and motility. Phosphorylation of Ack1 at tyrosine 284 is known to regulate its activity and function, making it a valuable biomarker for understanding the role of Ack1 in cancer, neurodegenerative diseases, and other disorders.
With the ACK1 Phospho-Tyr284 Colorimetric Cell-Based ELISA Kit, researchers can accurately assess the levels of phosphorylated Ack1 in cellular samples, allowing for in-depth investigations into Ack1-mediated signaling pathways and potential therapeutic targets. This kit is a valuable tool for advancing research in cell biology, cancer biology, and drug discovery.
| Product Name: | ACK1 (Phospho-Tyr284)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01332 |
| ELISA Type: | Cell-Based |
| Target: | ACK1 (Phospho-Tyr284) |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The ACK1 (Phospho-Tyr284) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ACK1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ACK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ACK1 phosphorylation.
Qualitative determination of ACK1 (Phospho-Tyr284) concentration is achieved by an indirect ELISA format. In essence, ACK1 (Phospho-Tyr284) is captured by ACK1 (Phospho-Tyr284)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 10188, UniProt ID: Q07912, OMIM: 606994, Unigene: Hs.518513 |
| Gene Symbol: | TNK2 |
| Sub Type: | Phospho |
| UniProt Protein Function: | Ack: a tyrosine kinase that binds Cdc42 in its GTP-bound form and inhibits both the intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activity of Cdc42. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain. May be involved in a regulatory mechanism that sustains the GTP-bound active form of Cdc42 and which is directly linked to a tyrosine phosphorylation signal transduction pathway. Amplification in primary tumors correlates with metastatic potential. Two differentially spliced isoforms have been described. |
| UniProt Protein Details: | Protein type:Kinase, protein; EC 2.7.11.1; EC 2.7.10.2; Protein kinase, tyrosine (non-receptor); Protein kinase, TK; TK group; Ack family Chromosomal Location of Human Ortholog: 3q29 Cellular Component: clathrin-coated vesicle; coated pit; cytoplasm; endosome; extrinsic to internal side of plasma membrane; membrane; nucleus; plasma membrane Molecular Function:epidermal growth factor receptor binding; GTPase inhibitor activity; identical protein binding; protein binding; protein serine/threonine/tyrosine kinase activity; protein-tyrosine kinase activity; WW domain binding Biological Process: cell differentiation; cell migration; cell surface receptor linked signal transduction; innate immune response; phosphorylation; positive regulation of peptidyl-tyrosine phosphorylation; regulation of cell proliferation; small GTPase mediated signal transduction; transmembrane receptor protein tyrosine kinase signaling pathway |
| NCBI Summary: | This gene encodes a tyrosine kinase that binds Cdc42Hs in its GTP-bound form and inhibits both the intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activity of Cdc42Hs. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain. The protein may be involved in a regulatory mechanism that sustains the GTP-bound active form of Cdc42Hs and which is directly linked to a tyrosine phosphorylation signal transduction pathway. Several alternatively spliced transcript variants have been identified from this gene, but the full-length nature of only two transcript variants has been determined. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q07912 |
| NCBI GenInfo Identifier: | 229462980 |
| NCBI Gene ID: | 10188 |
| NCBI Accession: | Q07912.3 |
| UniProt Secondary Accession: | Q07912,Q6ZMQ0, Q8N6U7, Q96H59, |
| UniProt Related Accession: | Q07912 |
| Molecular Weight: | 119,349 Da |
| NCBI Full Name: | Activated CDC42 kinase 1 |
| NCBI Synonym Full Names: | tyrosine kinase non receptor 2 |
| NCBI Official Symbol: | TNK2 |
| NCBI Official Synonym Symbols: | ACK; ACK1; ACK-1; p21cdc42Hs |
| NCBI Protein Information: | activated CDC42 kinase 1 |
| UniProt Protein Name: | Activated CDC42 kinase 1 |
| UniProt Synonym Protein Names: | Tyrosine kinase non-receptor protein 2 |
| Protein Family: | Activated CDC42 kinase |
| UniProt Gene Name: | TNK2 |
| UniProt Entry Name: | ACK1_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-ACK1 (Phospho-Tyr284) Antibody, Anti-ACK1 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.)
