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FRS2 (Phospho-Tyr436) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00501
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

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FRS2 (Phospho-Tyr436)Colorimetric Cell-Based ELISA Kit

The FRS2 Phospho-Tyr436 Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers looking to study the phosphorylation status of FRS2 at tyrosine 436 in cell-based assays. This kit provides a highly sensitive and accurate method for detecting and quantifying FRS2 phosphorylation levels in cell lysates, making it ideal for investigating signal transduction pathways and cell signaling mechanisms.FRS2 (Fibroblast Growth Factor Receptor Substrate 2) is a critical mediator of intracellular signaling cascades activated by growth factors such as FGF (Fibroblast Growth Factor). Phosphorylation of FRS2 at tyrosine 436 plays a key role in downstream signaling events, influencing cell growth, proliferation, and differentiation.

Dysregulation of FRS2 phosphorylation has been implicated in various diseases, including cancer and developmental disorders.With its high sensitivity and specificity, the FRS2 Phospho-Tyr436 Colorimetric Cell-Based ELISA Kit offers reliable and reproducible results, making it an essential tool for researchers studying FRS2 signaling pathways and developing potential targeted therapies. By accurately measuring FRS2 phosphorylation levels, this kit provides valuable insights into the molecular mechanisms governing cell behavior and disease progression.

Product Name:FRS2 (Phospho-Tyr436) Colorimetric Cell-Based ELISA
Product Code:CBCAB00501
ELISA Type:Cell-Based
Target:FRS2 (Phospho-Tyr436)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The FRS2 (Phospho-Tyr436) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect FRS2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated FRS2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on FRS2 phosphorylation.

Qualitative determination of FRS2 (Phospho-Tyr436) concentration is achieved by an indirect ELISA format. In essence, FRS2 (Phospho-Tyr436) is captured by FRS2 (Phospho-Tyr436)-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: 10818, UniProt ID: Q8WU20, OMIM: 607743, Unigene: Hs.593446
Gene Symbol:FRS2
Sub Type:Phospho
UniProt Protein Function:FRS2: an adaptor protein involved in fibroblast growth factor receptor (FGFR) signaling. Plays an important role in linking FGFR and nerve growth factor receptors with Ras/MAPK signaling pathways.
UniProt Protein Details:

Protein type:Adaptor/scaffold

Chromosomal Location of Human Ortholog: 12q15

Cellular Component: endosome; integral to plasma membrane; membrane; plasma membrane

Molecular Function:1-phosphatidylinositol-3-kinase activity; fibroblast growth factor receptor binding; neurotrophin TRKA receptor binding; phosphatase activator activity; phosphatidylinositol-4,5-bisphosphate 3-kinase activity; protein binding; Ras guanyl-nucleotide exchange factor activity; transmembrane receptor protein tyrosine kinase adaptor protein activity

Biological Process: activation of MAPKK activity; fibroblast growth factor receptor signaling pathway; G-protein coupled receptor protein signaling pathway; MAPKKK cascade; phosphoinositide-mediated signaling; regulation of phosphoinositide 3-kinase cascade; transmembrane receptor protein tyrosine phosphatase signaling pathway

UniProt Code:Q8WU20
NCBI GenInfo Identifier:209572769
NCBI Gene ID:10818
NCBI Accession:Q8WU20.4
UniProt Secondary Accession:Q8WU20,O43558, Q7LDQ6, B0LPF2, B2R684,
UniProt Related Accession:Q8WU20
Molecular Weight:57,029 Da
NCBI Full Name:Fibroblast growth factor receptor substrate 2
NCBI Synonym Full Names:fibroblast growth factor receptor substrate 2
NCBI Official Symbol:FRS2  
NCBI Official Synonym Symbols:SNT; SNT1; FRS1A; FRS2A; SNT-1; FRS2alpha  
NCBI Protein Information:fibroblast growth factor receptor substrate 2
UniProt Protein Name:Fibroblast growth factor receptor substrate 2
UniProt Synonym Protein Names:FGFR-signaling adaptor SNT; Suc1-associated neurotrophic factor target 1; SNT-1
Protein Family:Fibroblast growth factor receptor substrate
UniProt Gene Name:FRS2  
UniProt Entry Name:FRS2_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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-FRS2 (Phospho-Tyr436) Antibody, Anti-FRS2 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.)

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