Description
ICK (Phospho-Tyr159)Colorimetric Cell-Based ELISA Kit
The Ick (Immune Cell Kinase) phosphorylation at tyrosine 159 Colorimetric Cell-Based ELISA Kit is a powerful tool for analyzing specific phosphorylation events in immune cells. This kit allows for the quantitative detection of Ick phosphorylation at tyrosine 159 in cell lysates or tissue samples, providing valuable insights into immune cell signaling pathways.The phosphorylation of Ick at tyrosine 159 is known to regulate its activity and function in immune cells, making it a key target for research into immune response mechanisms and potential therapeutic interventions.
The Colorimetric Cell-Based ELISA Kit offers high sensitivity and specificity, ensuring accurate and reliable results for a wide range of experimental applications.Whether studying immune cell signaling in cancer, autoimmune diseases, or inflammatory conditions, the Ick phosphorylation at tyrosine 159 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers looking to deepen their understanding of immune cell biology and develop novel treatment strategies.
| Product Name: | ICK (Phospho-Tyr159)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01645 |
| ELISA Type: | Cell-Based |
| Target: | ICK (Phospho-Tyr159) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The ICK (Phospho-Tyr159) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ICK protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ICK in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ICK phosphorylation.
Qualitative determination of ICK (Phospho-Tyr159) concentration is achieved by an indirect ELISA format. In essence, ICK (Phospho-Tyr159) is captured by ICK (Phospho-Tyr159)-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: 22858, UniProt ID: Q9UPZ9, OMIM: 612325, Unigene: Hs.417022 |
| Gene Symbol: | ICK |
| Sub Type: | Phospho |
| UniProt Protein Function: | Required for ciliogenesis (PubMed:24797473). Phosphorylates KIF3A. Involved in the control of ciliary length (PubMed:24853502). Regulates the ciliary localization of SHH pathway components as well as the localization of IFT components at ciliary tips. May play a key role in the development of multiple organ systems and particularly in cardiac development. Regulates intraflagellar transport (IFT) speed and negatively regulates cilium length in a cAMP and mTORC1 signaling-dependent manner and this regulation requires its kinase activity. |
| NCBI Summary: | Eukaryotic protein kinases are enzymes that belong to a very extensive family of proteins which share a conserved catalytic core common with both serine/threonine and tyrosine protein kinases. This gene encodes an intestinal serine/threonine kinase harboring a dual phosphorylation site found in mitogen-activating protein (MAP) kinases. The protein localizes to the intestinal crypt region and is thought to be important in intestinal epithelial cell proliferation and differentiation. Alternative splicing has been observed at this locus and two variants, encoding the same isoform, have been identified. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q9UPZ9 |
| NCBI GenInfo Identifier: | 7662388 |
| NCBI Gene ID: | 22858 |
| NCBI Accession: | NP_055735.1 |
| UniProt Secondary Accession: | Q9UPZ9,O75985, Q5THL2, Q8IYH8, Q9BX17, Q9NYX3, A7MD41 |
| UniProt Related Accession: | Q9UPZ9 |
| Molecular Weight: | 71 kDa |
| NCBI Full Name: | serine/threonine-protein kinase ICK |
| NCBI Synonym Full Names: | intestinal cell kinase |
| NCBI Official Symbol: | ICK |
| NCBI Official Synonym Symbols: | ECO; MRK; LCK2 |
| NCBI Protein Information: | serine/threonine-protein kinase ICK |
| UniProt Protein Name: | Serine/threonine-protein kinase ICK |
| UniProt Synonym Protein Names: | Intestinal cell kinase; hICK; Laryngeal cancer kinase 2; LCK2; MAK-related kinase; MRK |
| Protein Family: | Serine/threonine-protein kinase |
| UniProt Gene Name: | ICK |
| 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-ICK (Phospho-Tyr159) Antibody, Anti-ICK 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.)
