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

Description

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 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.

How does our VEGFR2 (Phospho-Tyr1059) Fluorometric 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 Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. 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 RFU values.
2.	An antibody against the nonphosphorylated counterpart of VEGFR2 (Phospho-Tyr1059) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated VEGFR2 can be used to normalize the RFU value for phosphorylated VEGFR2.

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

Product Name:	VEGFR2 (Phospho-Tyr1059) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00036
Description:	The VEGFR2 (Phospho-Tyr1059) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor VEGFR2 (Phospho-Tyr1059) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated 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 KDR phosphorylation.
Dynamic Range:	> 5000 Cells
Detection Method:	Fluorometric
Storage/Stability:	4°C/6 Months
Reactivity:	Human, Mouse, Rat
Assay Type:	Cell-Based ELISA
Database Links:	Gene ID: 3791, UniProt ID: P35968, OMIM #: 191306, Unigene #: Hs.479756
Format:	Two 96-Well Plates
NCBI Gene Symbol:	KDR
Sub Type:	Phospho
Target Name:	Phospho-VEGFR2 (Tyr1059)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components	Quantity
96-Well Black Cell CultureClear-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	110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody	6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody	6 ml
Adhesive Plate Seals	2 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:

Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
Micropipettes capable of measuring volumes from 1 µl to 1 ml
Deionized or sterile water (ddH2O)
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)

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 desired cell concentration in culture medium into each well of the 96-well plates. 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 3 minutes. 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 3 minutes each time.
9.	Dispense 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 3 minutes each time.
11.	Add 50 µl of Primary Antibody Mixture P to corresponding wells for VEGFR2 (Phospho-Tyr1059) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total VEGFR2 detection. Cover the plate 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 3 minutes each time.
13.	Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.	Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.	Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

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