Interferon-gamma Receptor alpha (Phospho-Tyr457) Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB01430
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based Phospho Specific
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | Interferon-gamma Receptor alpha (Phospho-Tyr457) Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01430 |
| ELISA Type: | Cell-Based |
| Target: | Interferon-gamma Receptor alpha (Phospho-Tyr457) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The Interferon-gamma Receptor alpha (Phospho-Tyr457) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Interferon-gamma Receptor alpha protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Interferon-gamma Receptor alpha in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Interferon-gamma Receptor alpha phosphorylation.
Qualitative determination of Interferon-gamma Receptor alpha (Phospho-Tyr457) concentration is achieved by an indirect ELISA format. In essence, Interferon-gamma Receptor alpha (Phospho-Tyr457) is captured by Interferon-gamma Receptor alpha (Phospho-Tyr457)-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: 3459, UniProt ID: P15260, OMIM: 107470/209950/600263, Unigene: Hs.520414 |
| Gene Symbol: | IFNGR1 |
| Sub Type: | Phospho |
| UniProt Protein Function: | IFNGR1: Receptor for interferon gamma. Two receptors bind one interferon gamma dimer. Defects in IFNGR1 are a cause of mendelian susceptibility to mycobacterial disease (MSMD); also known as familial disseminated atypical mycobacterial infection. This rare condition confers predisposition to illness caused by moderately virulent mycobacterial species, such as Bacillus Calmette-Guerin (BCG) vaccine and environmental non-tuberculous mycobacteria, and by the more virulent Mycobacterium tuberculosis. Other microorganisms rarely cause severe clinical disease in individuals with susceptibility to mycobacterial infections, with the exception of Salmonella which infects less than 50% of these individuals. The pathogenic mechanism underlying MSMD is the impairment of interferon-gamma mediated immunity whose severity determines the clinical outcome. Some patients die of overwhelming mycobacterial disease with lepromatous-like lesions in early childhood, whereas others develop, later in life, disseminated but curable infections with tuberculoid granulomas. MSMD is a genetically heterogeneous disease with autosomal recessive, autosomal dominant or X-linked inheritance. Belongs to the type II cytokine receptor family. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Receptor, cytokine Chromosomal Location of Human Ortholog: 6q23.3 Cellular Component: integral to plasma membrane; endoplasmic reticulum; dendrite; postsynaptic density; plasma membrane; integral to membrane; vesicle Molecular Function:hematopoietin/interferon-class (D200-domain) cytokine receptor activity; protein binding; interferon-gamma receptor activity; cytokine binding Biological Process: cytokine and chemokine mediated signaling pathway; response to virus; signal transduction Disease: Helicobacter Pylori Infection, Susceptibility To; Immunodeficiency 27b; Immunodeficiency 27a; Mycobacterium Tuberculosis, Susceptibility To; Hepatitis B Virus, Susceptibility To |
| NCBI Summary: | This gene (IFNGR1) encodes the ligand-binding chain (alpha) of the gamma interferon receptor. Human interferon-gamma receptor is a heterodimer of IFNGR1 and IFNGR2. A genetic variation in IFNGR1 is associated with susceptibility to Helicobacter pylori infection. In addition, defects in IFNGR1 are a cause of mendelian susceptibility to mycobacterial disease, also known as familial disseminated atypical mycobacterial infection. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P15260 |
| NCBI GenInfo Identifier: | 124474 |
| NCBI Gene ID: | 3459 |
| NCBI Accession: | P15260.1 |
| UniProt Secondary Accession: | P15260,Q53Y96, B4DFT7, E1P587, |
| UniProt Related Accession: | P15260 |
| Molecular Weight: | Calculated: 21kDa/54kDaObserved: 70kDa |
| NCBI Full Name: | Interferon gamma receptor 1 |
| NCBI Synonym Full Names: | interferon gamma receptor 1 |
| NCBI Official Symbol: | IFNGR1 |
| NCBI Official Synonym Symbols: | CD119; IFNGR; IMD27A; IMD27B |
| NCBI Protein Information: | interferon gamma receptor 1; CDw119; AVP, type 2; IFN-gamma-R1; CD119 antigen; IFN-gamma receptor 1; antiviral protein, type 2; immune interferon receptor 1; interferon-gamma receptor alpha chain |
| UniProt Protein Name: | Interferon gamma receptor 1 |
| UniProt Synonym Protein Names: | CDw119; CD_antigen: CD119 |
| Protein Family: | Interferon gamma receptor |
| UniProt Gene Name: | IFNGR1 |
| UniProt Entry Name: | INGR1_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-Interferon-gamma Receptor alpha (Phospho-Tyr457) Antibody, Anti-Interferon-gamma Receptor alpha 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.)
