Description
GIRK1/KIR3.1/KCNJ3 (Phospho-Ser185)Colorimetric Cell-Based ELISA Kit
The Girk1/Kir3.1/KCNJ3 Phospho-Ser185 Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers seeking to study the phosphorylation of Girk1/Kir3.1/KCNJ3 at Ser185 in cell-based assays. This kit provides high sensitivity and specificity, allowing for accurate and reproducible results in a variety of experimental settings.The phosphorylation of Girk1/Kir3.1/KCNJ3 at Ser185 plays a crucial role in modulating the function of this protein complex, which is involved in regulating neuronal excitability and synaptic transmission. Dysregulation of Girk1/Kir3.
1/KCNJ3 phosphorylation has been implicated in various neurological disorders, making this kit an invaluable resource for researchers studying these conditions and potential therapeutic interventions.Overall, the Girk1/Kir3.1/KCNJ3 Phospho-Ser185 Colorimetric Cell-Based ELISA Kit offers a comprehensive solution for investigating the phosphorylation status of Girk1/Kir3.1/KCNJ3 in cellular models, providing valuable insights into the molecular mechanisms underlying neurological disorders and the development of targeted therapies.
| Product Name: | GIRK1/KIR3.1/KCNJ3 (Phospho-Ser185)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01445 |
| ELISA Type: | Cell-Based |
| Target: | GIRK1/KIR3.1/KCNJ3 (Phospho-Ser185) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The GIRK1/KIR3.1/KCNJ3 (Phospho-Ser185) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GIRK1/KIR3.1/KCNJ3 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated GIRK1/KIR3.1/KCNJ3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on GIRK1/KIR3.1/KCNJ3 phosphorylation.
Qualitative determination of GIRK1/KIR3.1/KCNJ3 (Phospho-Ser185) concentration is achieved by an indirect ELISA format. In essence, GIRK1/KIR3.1/KCNJ3 (Phospho-Ser185) is captured by GIRK1/KIR3.1/KCNJ3 (Phospho-Ser185)-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: 3760, UniProt ID: P48549, OMIM: 601534, Unigene: Hs.591606 |
| Gene Symbol: | KCNJ3 |
| Sub Type: | Phospho |
| UniProt Protein Function: | GIRK1: This potassium channel is controlled by G proteins. Inward rectifier potassium channels are characterized by a greater tendency to allow potassium to flow into the cell rather than out of it. Their voltage dependence is regulated by the concentration of extracellular potassium; as external potassium is raised, the voltage range of the channel opening shifts to more positive voltages. The inward rectification is mainly due to the blockage of outward current by internal magnesium. This receptor plays a crucial role in regulating the heartbeat. Belongs to the inward rectifier-type potassium channel (TC 1.A.2.1) family. KCNJ3 subfamily. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Membrane protein, multi-pass; Channel, potassium Chromosomal Location of Human Ortholog: 2q24.1 Cellular Component: external side of plasma membrane; plasma membrane; T-tubule; voltage-gated potassium channel complex Molecular Function:G-protein activated inward rectifier potassium channel activity; inward rectifier potassium channel activity; protein binding Biological Process: potassium ion import; potassium ion transport; response to electrical stimulus; synaptic transmission |
| NCBI Summary: | Potassium channels are present in most mammalian cells, where they participate in a wide range of physiologic responses. The protein encoded by this gene is an integral membrane protein and inward-rectifier type potassium channel. The encoded protein, which has a greater tendency to allow potassium to flow into a cell rather than out of a cell, is controlled by G-proteins and plays an important role in regulating heartbeat. It associates with three other G-protein-activated potassium channels to form a heteromultimeric pore-forming complex that also couples to neurotransmitter receptors in the brain and whereby channel activation can inhibit action potential firing by hyperpolarizing the plasma membrane. These multimeric G-protein-gated inwardly-rectifying potassium (GIRK) channels may play a role in the pathophysiology of epilepsy, addiction, Down's syndrome, ataxia, and Parkinson's disease. Alternative splicing results in multiple transcript variants encoding distinct proteins. [provided by RefSeq, May 2012] |
| UniProt Code: | P48549 |
| NCBI GenInfo Identifier: | 1352482 |
| NCBI Gene ID: | 3760 |
| NCBI Accession: | P48549.1 |
| UniProt Secondary Accession: | P48549,Q8TBI0, B4DEW7, |
| UniProt Related Accession: | P48549 |
| Molecular Weight: | 26,829 Da |
| NCBI Full Name: | G protein-activated inward rectifier potassium channel 1 |
| NCBI Synonym Full Names: | potassium voltage-gated channel subfamily J member 3 |
| NCBI Official Symbol: | KCNJ3 |
| NCBI Official Synonym Symbols: | KGA; GIRK1; KIR3.1 |
| NCBI Protein Information: | G protein-activated inward rectifier potassium channel 1 |
| UniProt Protein Name: | G protein-activated inward rectifier potassium channel 1 |
| UniProt Synonym Protein Names: | Inward rectifier K(+) channel Kir3.1; Potassium channel, inwardly rectifying subfamily J member 3 |
| Protein Family: | G protein-activated inward rectifier potassium channel |
| UniProt Gene Name: | KCNJ3 |
| UniProt Entry Name: | KCNJ3_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-GIRK1/KIR3.1/KCNJ3 (Phospho-Ser185) Antibody, Anti-GIRK1/KIR3.1/KCNJ3 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.)
