Mouse Vascular endothelial growth factor receptor 2 (Kdr) ELISA Kit (MOEB0141)

Product Name:	Mouse Vascular endothelial growth factor receptor 2 (Kdr) ELISA Kit
SKU:	MOEB0141
Size:	96T
Target:	Mouse Vascular endothelial growth factor receptor 2 (Kdr)
Synonyms:	Fetal liver kinase 1, Kinase NYK, Protein-tyrosine kinase receptor flk-1, FLK-1, CD309, VEGFR-2, Flk-1, Flk1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	0.312-20ng/mL
Sensitivity:	0.178ng/mL

Intra CV:

4.6%

Inter CV:

9.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	100-109%	106-116%	87-97%	85-94%
EDTA Plasma(N=5)	103-114%	100-110%	105-114%	85-95%
Heparin Plasma(N=5)	100-112%	89-101%	109-119%	105-114%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	96	90-102
Plasma	98	92-104

Function:

Tyrosine-protein kinase that acts as a cell-surface receptor for VEGFA, VEGFC and VEGFD. Plays an essential role in the regulation of angiogenesis, vascular development, vascular permeability, and embryonic hematopoiesis. Promotes proliferation, survival, migration and differentiation of endothelial cells. Promotes reorganization of the actin cytoskeleton. Isoforms lacking a transmembrane domain, such as isoform 2, may function as decoy receptors for VEGFA, VEGFC and/or VEGFD. Isoform 2 plays an important role as a negative regulator of VEGFA- and VEGFC-mediated lymphangiogenesis by limiting the amount of free VEGFA and/or VEGFC and by preventing their binding to FLT4. Modulates FLT1 and FLT4 signaling by forming heterodimers. Binding of vascular growth factors to isoform 1 leads to the activation of several signaling cascades. Activation of PLCG1 leads to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate and the activation of protein kinase C. Mediates activation of MAPK1/ERK2, MAPK3/ERK1 and the MAP kinase signaling pathway, as well as of the AKT1 signaling pathway. Mediates phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3-kinase, reorganization of the actin cytoskeleton and activation of PTK2/FAK1. Required for VEGFA-mediated induction of NOS2 and NOS3, leading to the production of the signaling molecule nitric oxide (NO) by endothelial cells. Phosphorylates PLCG1. Promotes phosphorylation of FYN, NCK1, NOS3, PIK3R1, PTK2/FAK1 and SRC.

Uniprot:	P35918
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse Vascular endothelial growth factor receptor 2
Sub Unit:	Homodimer in the presence of bound dimeric VEGFA, VEGFC or VEGFD ligands; monomeric in the absence of bound ligands. Can also form heterodimers with FLT1/VEGFR1 and FLT4/VEGFR2. Interacts (tyrosine phosphorylated) with LFYN, NCK1, PLCG1. Interacts (tyrosine-phosphorylated active form preferentially) with DAB2IP (via C2 domain and active form preferentially); the interaction occurs at the late phase of VEGFA response and inhibits KDR/VEGFR2 activity. Interacts with SHBSH2D2A/TSAD, GRB2, MYOF, CBL and PDCD6 (PubMed:12796773, PubMed:12881528, PubMed:15026417, PubMed:15673613, PubMed:17702744, PubMed:19668192, PubMed:7681362, PubMed:8356051). Interacts (via C-terminus domain) with ERN1 (via kinase domain); the interaction is facilitated in a XBP1 isoform 1- and vascular endothelial growth factor (VEGF)-dependent manner in endothelial cells.
Subcellular Location:	Isoform 2 Secreted
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.