VEGFR2 (Phospho-Tyr1059) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00045
€599

Description

VEGFR2 (Phospho-Tyr1059) Cell-Based ELISA Kit

The VEGFR2 (Phospho-Tyr1059) Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor VEGFR2 (Phospho-Tyr1059) protein expression profile in cells. The kit can be used for measuring the relative amounts of VEGFR2 (Phospho-Tyr1059) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on VEGFR2 phosphorylation.

How does our VEGFR2 (Phospho-Tyr1059) Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of VEGFR2 (Phospho-Tyr1059) concentration is achieved by an indirect ELISA format. In essence, VEGFR2 (Phospho-Tyr1059) is captured by VEGFR2 (Phospho-Tyr1059) -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° 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.

VEGFR2 (Phospho-Tyr1059) Colorimetric Cell-Based ELISA Kit -Information

Product Name:VEGFR2 (Phospho-Tyr1059) Colorimetric Cell-Based ELISA Kit
Product Code/SKU:CBCAB00045
Description:The VEGFR2 (Phospho-Tyr1059) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect VEGFR2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated VEGFR2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on VEGFR2 phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based
Format:2 x 96-Well Microplates
Database Links:Gene ID: 3791, UniProt ID: P35968, OMIM #: 191306, Unigene #: Hs.479756
NCBI Gene Symbol:KDR
Sub Type:Phospho
Target Name:VEGFR2 (Phospho-Tyr1059)

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

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 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
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

Additional equipment and materials required

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 ?l 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

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

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-VEGFR2 (Phospho-Tyr1059) Antibody, Anti-VEGFR2 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.)

VEGFR2 (Phospho-Tyr1059) - Protein Information

UniProt Protein Function:VEGFR2: a receptor tyrosine kinase of the VEGFR family. High affinity receptor for VEGF and VEGF-C. Ligand binding induces autophosphorylation and activation. Activated receptor recruits proteins including Shc, GRB2, PI3K, Nck, SHP-1 and SHP-2. Plays a key role in vascular development and regulation of vascular permeability.
UniProt Protein Details:

Protein type:Protein kinase, TK; Kinase, protein; Membrane protein, integral; EC 2.7.10.1; Protein kinase, tyrosine (receptor); TK group; VEGFR family

Chromosomal Location of Human Ortholog: 4q11-q12

Cellular Component: Golgi apparatus; endoplasmic reticulum; integral to plasma membrane; early endosome; cytoplasmic membrane-bound vesicle; extracellular region; plasma membrane; cell junction; nucleus; endosome; lipid raft; external side of plasma membrane

Molecular Function:integrin binding; vascular endothelial growth factor receptor activity; protein binding; growth factor binding; protein-tyrosine kinase activity; receptor signaling protein tyrosine kinase activity; Hsp90 protein binding; transmembrane receptor protein tyrosine kinase activity; ATP binding

Biological Process: extracellular matrix organization and biogenesis; positive regulation of positive chemotaxis; peptidyl-tyrosine phosphorylation; viral reproduction; protein amino acid autophosphorylation; cell maturation; calcium ion homeostasis; regulation of cell shape; positive regulation of MAPKKK cascade; positive regulation of focal adhesion formation; ovarian follicle development; positive regulation of cell proliferation; positive regulation of mesenchymal cell proliferation; angiogenesis; vasculogenesis; endothelial cell differentiation; cell fate commitment; embryonic hemopoiesis; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of angiogenesis; cell migration during sprouting angiogenesis; branching morphogenesis of a tube; positive regulation of endothelial cell proliferation; positive regulation of protein amino acid phosphorylation; lymph vessel development; vascular endothelial growth factor receptor signaling pathway; alveolus development; surfactant homeostasis; transmembrane receptor protein tyrosine kinase signaling pathway; negative regulation of apoptosis; positive regulation of cell migration; positive regulation of nitric-oxide synthase biosynthetic process

Disease: Hemangioma, Capillary Infantile

NCBI Summary:Vascular endothelial growth factor (VEGF) is a major growth factor for endothelial cells. This gene encodes one of the two receptors of the VEGF. This receptor, known as kinase insert domain receptor, is a type III receptor tyrosine kinase. It functions as the main mediator of VEGF-induced endothelial proliferation, survival, migration, tubular morphogenesis and sprouting. The signalling and trafficking of this receptor are regulated by multiple factors, including Rab GTPase, P2Y purine nucleotide receptor, integrin alphaVbeta3, T-cell protein tyrosine phosphatase, etc.. Mutations of this gene are implicated in infantile capillary hemangiomas. [provided by RefSeq, May 2009]
UniProt Code:P35968
NCBI GenInfo Identifier:9087218
NCBI Gene ID:3791
NCBI Accession:P35968.2
UniProt Secondary Accession:P35968,O60723, Q14178, A2RRS0, B5A925, C5IFA0,
UniProt Related Accession:P35968
Molecular Weight:79,634 Da
NCBI Full Name:Vascular endothelial growth factor receptor 2
NCBI Synonym Full Names:kinase insert domain receptor (a type III receptor tyrosine kinase)
NCBI Official Symbol:KDR  
NCBI Official Synonym Symbols:FLK1; CD309; VEGFR; VEGFR2  
NCBI Protein Information:vascular endothelial growth factor receptor 2; soluble VEGFR2; fetal liver kinase 1; fetal liver kinase-1; protein-tyrosine kinase receptor Flk-1; tyrosine kinase growth factor receptor
UniProt Protein Name:Vascular endothelial growth factor receptor 2
UniProt Synonym Protein Names:Fetal liver kinase 1; FLK-1; Kinase insert domain receptor; KDR; Protein-tyrosine kinase receptor flk-1; CD_antigen: CD309
Protein Family:Vascular endothelial growth factor receptor
UniProt Gene Name:KDR  
UniProt Entry Name:VGFR2_HUMAN
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