Mouse VEGFR3/FLT4 ELISA Kit (MOFI00112)

Product Name:	Mouse VEGFR3/FLT4 ELISA Kit
Product Code:	MOFI00112
Size:	96 Assays
Alias:	FLT4, VEGF R3, FLT-4, LMPH1A, PCLFLT41, VEGFR-3, VEGFR3, fms-related tyrosine kinase 4, soluble VEGFR3 variant 1, soluble VEGFR3 variant 2, soluble VEGFR3 variant 3, Tyrosine-protein kinase receptor FLT4, vascular endothelial growth factor receptor 3, Fms-like tyrosine kinase 4
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse VEGFR3/FLT4R3/FLT4R3/FLT4 concentrations in serum plasma and other biological fluids.
Sensitivity:	37.5pg/ml
Range:	62.5-4000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse VEGFR3/FLT4R3/FLT4R3/FLT4 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse VEGFR3/FLT4R3/FLT4R3/FLT4 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-99	95
EDTA plasma(n=5)	89-103	95
UFH plasma(n=5)	89-101	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse VEGFR3/FLT4R3/FLT4R3/FLT4 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)	87-99%	94-105%	85-104%
EDTA plasma(n=5)	82-101%	89-94%	85-91%
UFH plasma(n=5)	85-99%	80-100%	81-90%

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

P35917

UniProt Protein Function:	VEGFR3: Tyrosine-protein kinase that acts as a cell-surface receptor for VEGFC and VEGFD, and plays an essential role in adult lymphangiogenesis and in the development of the vascular network and the cardiovascular system during embryonic development. Promotes proliferation, survival and migration of endothelial cells, and regulates angiogenic sprouting. Signaling by activated FLT4 leads to enhanced production of VEGFC, and to a lesser degree VEGFA, thereby creating a positive feedback loop that enhances FLT4 signaling. Modulates KDR signaling by forming heterodimers. The secreted isoform 3 may function as a decoy receptor for VEGFC and/or VEGFD and play an important role as a negative regulator of VEGFC-mediated lymphangiogenesis and angiogenesis. Binding of vascular growth factors to isoform 1 or isoform 2 leads to the activation of several signaling cascades; isoform 2 seems to be less efficient in signal transduction, because it has a truncated C-terminus and therefore lacks several phosphorylation sites. Mediates activation of the MAPK1/ERK2, MAPK3/ERK1 signaling pathway, of MAPK8 and the JUN signaling pathway, and of the AKT1 signaling pathway. Phosphorylates SHC1. Mediates phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3- kinase. Promotes phosphorylation of MAPK8 at 'Thr-183' and 'Tyr- 185', and of AKT1 at 'Ser-473'. Interacts with VEGFC and VEGFD. Monomer in the absence of bound VEGFC or VEGFD. Homodimer in the presence of bound VEGFC or VEGFD. Can also form a heterodimer with KDR. Interacts with PTPN14; the interaction is enhanced by stimulation with VEGFC. Interacts with CRK, GRB2, PTK2/FAK1, SHC1, PIK3R1 and PTPN11/SHP- 2. Identified in a complex with SRC and ITGB1. Detected in endothelial cells. Widely expressed. Detected in fetal spleen, lung and brain. Detected in adult liver, muscle, thymus, placenta, lung, testis, ovary, prostate, heart, and kidney. Present in an inactive conformation in the absence of bound ligand. Binding of VEGFC or VEGFD leads to dimerization and activation by autophosphorylation on tyrosine residues. Inhibited by MAZ51. Belongs to the protein kinase superfamily. Tyr protein kinase family. CSF-1/PDGF receptor subfamily. 3 isoforms of the human protein are produced by alternative splicing.
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 Cellular Component: cytoplasm; integral to membrane; integral to plasma membrane; membrane; nucleus; plasma membrane; receptor complex Molecular Function:ATP binding; growth factor binding; kinase activity; nucleotide binding; protein binding; protein kinase activity; protein phosphatase binding; protein-tyrosine kinase activity; transferase activity; transmembrane receptor protein tyrosine kinase activity; vascular endothelial growth factor receptor activity Biological Process: alveolus development; angiogenesis; blood vessel morphogenesis; lymph vessel development; lymphangiogenesis; negative regulation of apoptosis; peptidyl-tyrosine phosphorylation; phosphorylation; positive regulation of cell growth; positive regulation of cell proliferation; positive regulation of endothelial cell proliferation; positive regulation of JNK cascade; positive regulation of MAPKKK cascade; positive regulation of protein amino acid phosphorylation; protein amino acid autophosphorylation; protein amino acid phosphorylation; respiratory gaseous exchange; respiratory system process; sprouting angiogenesis; transmembrane receptor protein tyrosine kinase signaling pathway; vascular endothelial growth factor receptor signaling pathway; vasculature development
UniProt Code:	P35917
NCBI GenInfo Identifier:	544322
NCBI Gene ID:	14257
NCBI Accession:	P35917.1
UniProt Related Accession:	P35917
Molecular Weight:	153,016 Da
NCBI Full Name:	Vascular endothelial growth factor receptor 3
NCBI Synonym Full Names:	FMS-like tyrosine kinase 4
NCBI Official Symbol:	Flt4
NCBI Official Synonym Symbols:	Chy; Flt-4; VEGFR3; VEGFR-3; AI323512
NCBI Protein Information:	vascular endothelial growth factor receptor 3
UniProt Protein Name:	Vascular endothelial growth factor receptor 3
UniProt Synonym Protein Names:	Fms-like tyrosine kinase 4; FLT-4; Tyrosine-protein kinase receptor FLT4
Protein Family:	Vascular endothelial growth factor receptor
UniProt Gene Name:	Flt4
UniProt Entry Name:	VGFR3_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 VEGFR3/FLT4 ELISA Kit (MOFI00112)

Description