Mouse VEGF R2/KDR ELISA Kit (MOFI01185)

Product Name:	Mouse VEGF R2/KDR ELISA Kit
Product Code:	MOFI01185
Size:	96 Assays
Alias:	VEGFR-2, KDR, Vascuoar Endothelial Growth Factor Receptor 2, VEGFR2, VEGFR, CD309, Flk-1, CD309 antigen, Fetal liver kinase 1, fetal liver kinase-1, FLK-1, FLK1, tyrosine kinase growth factor receptor, Kinase insert domain receptor, kinase insert domain receptor, a type III receptor tyrosine kinase, Protein-tyrosine kinase receptor flk-1, soluble VEGFR2
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse VEGFR-2/KDR concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse VEGFR-2/KDR and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse VEGFR-2/KDR in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-99	93
EDTA plasma(n=5)	92-105	98
UFH plasma(n=5)	85-99	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse VEGFR-2/KDR and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-98%	85-98%	88-104%
EDTA plasma(n=5)	83-101%	83-99%	85-101%
UFH plasma(n=5)	83-99%	82-92%	82-100%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P35918

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, tyrosine (receptor); Kinase, protein; EC 2.7.10.1; Protein kinase, TK; Membrane protein, integral; TK group; VEGFR family Cellular Component: Golgi apparatus; neuron projection; cell surface; endoplasmic reticulum; integral to plasma membrane; early endosome; extracellular region; integral to membrane; cytosol; lipid raft; membrane; cell soma; cytoplasm; plasma membrane; cytoplasmic vesicle; nucleus; cell junction; endosome; external side of plasma membrane Molecular Function:integrin binding; transferase activity; vascular endothelial growth factor receptor activity; protein binding; protein-tyrosine kinase activity; growth factor binding; transferase activity, transferring phosphorus-containing groups; nucleotide binding; kinase activity; transmembrane receptor protein tyrosine kinase activity; ATP binding; protein kinase activity Biological Process: positive regulation of positive chemotaxis; multicellular organismal development; cell maturation; positive regulation of vasodilation; positive regulation of long-term neuronal synaptic plasticity; protein amino acid phosphorylation; calcium ion homeostasis; regulation of cell shape; elevation of cytosolic calcium ion concentration; positive regulation of mesenchymal cell proliferation; negative regulation of neuron apoptosis; positive regulation of TOR signaling pathway; response to drug; regulation of endothelial cell proliferation; cell fate commitment; embryonic hemopoiesis; regulation of endothelial cell differentiation; 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; lymph vessel development; surfactant homeostasis; alveolus development; transmembrane receptor protein tyrosine kinase signaling pathway; positive regulation of epithelial cell proliferation; positive regulation of nitric-oxide synthase biosynthetic process; negative regulation of apoptosis; peptidyl-tyrosine phosphorylation; protein amino acid autophosphorylation; positive regulation of calcium-mediated signaling; positive regulation of vascular endothelial growth factor receptor signaling pathway; positive regulation of MAPKKK cascade; positive regulation of focal adhesion formation; ovarian follicle development; positive regulation of cell proliferation; hemopoiesis; angiogenesis; vasculogenesis; cell differentiation; positive regulation of BMP signaling pathway; endothelial cell differentiation; cell migration; male gonad development; response to hypoxia; positive regulation of protein amino acid phosphorylation; vascular endothelial growth factor receptor signaling pathway; phosphorylation; neurite morphogenesis; positive regulation of cell migration; lung development
UniProt Code:	P35918
NCBI GenInfo Identifier:	549321
NCBI Gene ID:	16542
NCBI Accession:	P35918.1
UniProt Secondary Accession:	P35918,C5H7S5,
UniProt Related Accession:	P35918
Molecular Weight:	75,230 Da
NCBI Full Name:	Vascular endothelial growth factor receptor 2
NCBI Synonym Full Names:	kinase insert domain protein receptor
NCBI Official Symbol:	Kdr
NCBI Official Synonym Symbols:	Flk1; Ly73; Flk-1; Krd-1; VEGFR2; VEGFR-2; sVEGFR-2; 6130401C07
NCBI Protein Information:	vascular endothelial growth factor receptor 2; kinase NYK; VEGF receptor-2; fetal liver kinase 1; protein-tyrosine kinase receptor flk-1; vascular endothelial growth factor receptor-2; vascular endothelial growth factor receptor-3; vascular endothelial growth factor receptor- 2; soluble vascular endothelial growth factor receptor 2
UniProt Protein Name:	Vascular endothelial growth factor receptor 2
UniProt Synonym Protein Names:	Fetal liver kinase 1; FLK-1; Kinase NYK; Protein-tyrosine kinase receptor flk-1; CD_antigen: CD309
Protein Family:	Vascular endothelial growth factor receptor
UniProt Gene Name:	Kdr
UniProt Entry Name:	VGFR2_MOUSE

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum:	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid:	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant:	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates:	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue homogenates:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates:	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk:	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Mouse VEGF R2/KDR ELISA Kit (MOFI01185)

Description