Product Name:	Mouse VEGFB ELISA Kit
Product Code:	MOFI00121
Size:	96 Assays
Alias:	VEGF-B, Vascular Endothelial cell Growth Factor B
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse VEGFB concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-97	92
EDTA plasma(n=5)	85-103	91
UFH plasma(n=5)	89-104	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse VEGFB 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)	87-92%	85-94%	85-95%
EDTA plasma(n=5)	83-95%	87-98%	90-101%
UFH plasma(n=5)	86-99%	86-99%	81-96%

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

P49766

UniProt Protein Function:	VEGF: Growth factor active in angiogenesis, vasculogenesis and endothelial cell growth. Induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis and induces permeabilization of blood vessels. Binds to the FLT1/VEGFR1 and KDR/VEGFR2 receptors, heparan sulfate and heparin. NRP1/Neuropilin-1 binds isoforms VEGF-165 and VEGF-145. Isoform VEGF165B binds to KDR but does not activate downstream signaling pathways, does not activate angiogenesis and inhibits tumor growth. Defects in VEGFA are a cause of susceptibility to microvascular complications of diabetes type 1 (MVCD1). These are pathological conditions that develop in numerous tissues and organs as a consequence of diabetes mellitus. They include diabetic retinopathy, diabetic nephropathy leading to end-stage renal disease, and diabetic neuropathy. Diabetic retinopathy remains the major cause of new-onset blindness among diabetic adults. It is characterized by vascular permeability and increased tissue ischemia and angiogenesis. Belongs to the PDGF/VEGF growth factor family. 13 isoforms of the human protein are produced by alternative promoter.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Cytokine; Secreted; Secreted, signal peptide Cellular Component: extracellular space; cell surface; membrane; cytoplasm; plasma membrane; extracellular region; basement membrane; secretory granule Molecular Function:heparin binding; identical protein binding; protein homodimerization activity; growth factor activity; extracellular matrix binding; cytokine activity; platelet-derived growth factor receptor binding; vascular endothelial growth factor receptor 1 binding; vascular endothelial growth factor receptor binding; receptor agonist activity; protein binding; vascular endothelial growth factor receptor 2 binding; protein heterodimerization activity; fibronectin binding; receptor binding; chemoattractant activity Biological Process: heart morphogenesis; positive regulation of cell adhesion; macrophage differentiation; positive regulation of positive chemotaxis; multicellular organismal development; cell maturation; positive regulation of receptor internalization; basophil chemotaxis; regulation of cell shape; positive regulation of MAP kinase activity; positive chemotaxis; positive regulation of mesenchymal cell proliferation; mesoderm development; negative regulation of neuron apoptosis; kidney development; positive regulation of neuroblast proliferation; nervous system development; T-helper 1 type immune response; positive regulation of signal transduction; monocyte differentiation; mRNA stabilization; positive regulation of blood vessel endothelial cell migration; activation of CREB transcription factor; positive regulation of protein amino acid autophosphorylation; positive regulation of vascular permeability; regulation of endothelial cell differentiation; patterning of blood vessels; regulation of transcription from RNA polymerase II promoter; eye photoreceptor cell development; positive regulation of angiogenesis; positive regulation of peptidyl-tyrosine phosphorylation; camera-type eye morphogenesis; branching morphogenesis of a tube; cell migration during sprouting angiogenesis; cardiac muscle fiber development; positive regulation of cell division; positive regulation of axon extension involved in axon guidance; activation of protein kinase activity; blood vessel morphogenesis; endothelial cell migration; neuron development; positive regulation of transcription from RNA polymerase II promoter; positive regulation of endothelial cell proliferation; regulation of cGMP metabolic process; surfactant homeostasis; alveolus development; positive regulation of epithelial cell proliferation; negative regulation of apoptosis; lactation; post-embryonic camera-type eye development; positive regulation of smooth muscle cell proliferation; negative regulation of caspase activity; negative regulation of transcription from RNA polymerase II promoter; positive regulation of vascular endothelial growth factor receptor signaling pathway; induction of positive chemotaxis; positive regulation of focal adhesion formation; epithelial cell differentiation; ovarian follicle development; vasculature development; lymphangiogenesis; positive regulation of cell proliferation; negative regulation of programmed cell death; angiogenesis; cell differentiation; negative regulation of bone resorption; blood vessel development; cell migration; in utero embryonic development; lumen formation; positive regulation of cell motility; positive regulation of peptidyl-serine phosphorylation; positive regulation of protein kinase B signaling cascade; cell proliferation; positive regulation of protein complex assembly; response to hypoxia; artery morphogenesis; blood vessel remodeling; negative regulation of cell-cell adhesion; positive regulation of protein amino acid phosphorylation; sprouting angiogenesis; vascular endothelial growth factor receptor signaling pathway; lung development; growth; positive regulation of cell migration
NCBI Summary:	This gene is a member of the PDGF/VEGF growth factor family. It encodes a heparin-binding protein, which exists as a disulfide-linked homodimer. This growth factor induces proliferation and migration of vascular endothelial cells, and is essential for both physiological and pathological angiogenesis. Disruption of this gene in mice resulted in abnormal embryonic blood vessel formation. This gene is upregulated in many known tumors and its expression is correlated with tumor stage and progression. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. There is also evidence for alternative translation initiation from upstream non-AUG (CUG) codons resulting in additional isoforms. A recent study showed that a C-terminally extended isoform is produced by use of an alternative in-frame translation termination codon via a stop codon readthrough mechanism, and that this isoform is antiangiogenic. Expression of some isoforms derived from the AUG start codon is regulated by a small upstream open reading frame, which is located within an internal ribosome entry site.[provided by RefSeq, Nov 2015]
UniProt Code:	P49766
NCBI GenInfo Identifier:	160358799
NCBI Gene ID:	22339
NCBI Accession:	NP_001020421.2
UniProt Secondary Accession:	P49766,P49766,
UniProt Related Accession:	Q00731
Molecular Weight:
NCBI Full Name:	vascular endothelial growth factor A isoform 1
NCBI Synonym Full Names:	vascular endothelial growth factor A
NCBI Official Symbol:	Vegfa
NCBI Official Synonym Symbols:	Vpf; Vegf
NCBI Protein Information:	vascular endothelial growth factor A
UniProt Protein Name:	Vascular endothelial growth factor A
UniProt Synonym Protein Names:	Vascular permeability factor; VPF
Protein Family:	VEGF coregulated chemokine
UniProt Gene Name:	Vegfa
UniProt Entry Name:	VEGFA_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.