Description
FGFR2 Colorimetric Cell-Based ELISA Kit
The FGFR2 Colorimetric Cell-Based ELISA Kit from Assay Genie is a comprehensive solution for the quantitative measurement of Fibroblast Growth Factor Receptor 2 (FGFR2) levels in cell samples. This kit offers high sensitivity and accuracy, allowing for precise and reliable results in a variety of research applications.FGFR2 is a key receptor involved in cell growth, differentiation, and tissue repair, making it a critical target for studying various diseases, including cancer and developmental disorders. The FGFR2 Colorimetric Cell-Based ELISA Kit provides researchers with a valuable tool for investigating the role of FGFR2 in disease pathogenesis and evaluating potential therapeutic interventions.
With its user-friendly protocol and robust performance, the FGFR2 Colorimetric Cell-Based ELISA Kit is an essential resource for researchers seeking to explore the functional significance of FGFR2 in cell signaling pathways and disease progression. Trust Assay Genie for high-quality, reliable tools for your research needs.
| Product Name: | FGFR2 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00657 |
| ELISA Type: | Cell-Based |
| Target: | FGFR2 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The FGFR2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect FGFR2 protein expression profile in cells. The kit can be used for measuring the relative amounts of FGFR2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on FGFR2.
Qualitative determination of FGFR2 concentration is achieved by an indirect ELISA format. In essence, FGFR2 is captured by FGFR2-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: 2263, UniProt ID: P21802, OMIM: 176943, Unigene: Hs.533683 |
| Gene Symbol: | FGFR2 |
| Sub Type: | None |
| UniProt Protein Function: | FGFR2: a receptor tyrosine kinase of the highly-conserved FGFR family that binds fibroblast growth factor (FGF). Mutations are associated with many craniosynostotic syndromes and bone malformations. Mutations cause syndromes with defects in facial and limb development, including Crouzon syndrome, Beare-Stevenson cutis gyrata syndrome, Pfeiffer syndrome, Apert syndrome, and Jackson-Weiss syndrome. Somatic mutations seen in gastric cancer. Amplified in gastric, breast and some B cell cancers, but deleted in glioblastoma Twenty splice-variant isoforms have been described. |
| UniProt Protein Details: | Protein type:EC 2.7.10.1; FGFR family; Kinase, protein; Membrane protein, integral; Oncoprotein; Protein kinase, TK; Protein kinase, tyrosine (receptor); TK group Chromosomal Location of Human Ortholog: 10q26.13 Cellular Component: cell cortex; cell surface; cytoplasm; excitatory synapse; extracellular matrix; integral to plasma membrane; intracellular membrane-bound organelle; nucleoplasm; nucleus; plasma membrane Molecular Function:1-phosphatidylinositol-3-kinase activity; fibroblast growth factor binding; fibroblast growth factor receptor activity; phosphatidylinositol-4,5-bisphosphate 3-kinase activity; protein binding; protein homodimerization activity; protein-tyrosine kinase activity; Ras guanyl-nucleotide exchange factor activity Biological Process: alveolus development; angiogenesis; axonogenesis; bone mineralization; branching morphogenesis of a nerve; cell fate commitment; cell-cell signaling; embryonic cranial skeleton morphogenesis; embryonic digestive tract morphogenesis; embryonic organ development; embryonic organ morphogenesis; embryonic pattern specification; epidermis morphogenesis; epithelial cell differentiation; fibroblast growth factor receptor signaling pathway; gland morphogenesis; gut development; hair follicle morphogenesis; in utero embryonic development; inner ear morphogenesis; lacrimal gland development; limb bud formation; lung development; MAPKKK cascade; mesenchymal cell differentiation; midbrain development; morphogenesis of embryonic epithelium; multicellular organism growth; negative regulation of transcription from RNA polymerase II promoter; neuroblast division in the ventricular zone; odontogenesis; orbitofrontal cortex development; organ growth; organ morphogenesis; otic vesicle formation; peptidyl-tyrosine phosphorylation; phosphoinositide-mediated signaling; positive regulation of cardiac muscle cell proliferation; positive regulation of cell cycle; positive regulation of cell division; positive regulation of cell proliferation; positive regulation of epithelial cell proliferation; positive regulation of MAPKKK cascade; positive regulation of mesenchymal cell proliferation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of Wnt receptor signaling pathway; post-embryonic development; protein amino acid autophosphorylation; pyramidal neuron development; regulation of cell fate commitment; regulation of fibroblast growth factor receptor signaling pathway; regulation of multicellular organism growth; regulation of osteoblast differentiation; regulation of osteoblast proliferation; regulation of phosphoinositide 3-kinase cascade; regulation of smooth muscle cell differentiation; regulation of smoothened signaling pathway; reproductive structure development; skeletal morphogenesis; ureteric bud development; ventricular cardiac muscle morphogenesis Disease: Antley-bixler Syndrome Without Genital Anomalies Or Disordered Steroidogenesis; Apert Syndrome; Beare-stevenson Cutis Gyrata Syndrome; Bent Bone Dysplasia Syndrome; Crouzon Syndrome; Gastric Cancer; Jackson-weiss Syndrome; Lacrimoauriculodentodigital Syndrome; Pfeiffer Syndrome; Saethre-chotzen Syndrome; Scaphocephaly, Maxillary Retrusion, And Mental Retardation |
| NCBI Summary: | The protein encoded by this gene is a member of the fibroblast growth factor receptor family, where amino acid sequence is highly conserved between members and throughout evolution. FGFR family members differ from one another in their ligand affinities and tissue distribution. A full-length representative protein consists of an extracellular region, composed of three immunoglobulin-like domains, a single hydrophobic membrane-spanning segment and a cytoplasmic tyrosine kinase domain. The extracellular portion of the protein interacts with fibroblast growth factors, setting in motion a cascade of downstream signals, ultimately influencing mitogenesis and differentiation. This particular family member is a high-affinity receptor for acidic, basic and/or keratinocyte growth factor, depending on the isoform. Mutations in this gene are associated with Crouzon syndrome, Pfeiffer syndrome, Craniosynostosis, Apert syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrata syndrome, Saethre-Chotzen syndrome, and syndromic craniosynostosis. Multiple alternatively spliced transcript variants encoding different isoforms have been noted for this gene. [provided by RefSeq, Jan 2009] |
| UniProt Code: | P21802 |
| NCBI GenInfo Identifier: | 120049 |
| NCBI Gene ID: | 2263 |
| NCBI Accession: | P21802.1 |
| UniProt Secondary Accession: | P21802,P18443, Q01742, Q12922, Q14300, Q14301, Q14302 Q14303, Q14304, B4DFC2, E7EVR6, E9PCR0, |
| UniProt Related Accession: | P21802 |
| Molecular Weight: | 79,833 Da |
| NCBI Full Name: | Fibroblast growth factor receptor 2 |
| NCBI Synonym Full Names: | fibroblast growth factor receptor 2 |
| NCBI Official Symbol: | FGFR2 |
| NCBI Official Synonym Symbols: | BEK; JWS; BBDS; CEK3; CFD1; ECT1; KGFR; TK14; TK25; BFR-1; CD332; K-SAM |
| NCBI Protein Information: | fibroblast growth factor receptor 2 |
| UniProt Protein Name: | Fibroblast growth factor receptor 2 |
| UniProt Synonym Protein Names: | K-sam; KGFR; Keratinocyte growth factor receptor; CD_antigen: CD332 |
| Protein Family: | Tumor necrosis factor receptor superfamily |
| UniProt Gene Name: | FGFR2 |
| 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-FGFR2 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.)
