Human VEGFR1 / Flt-1 ELISA Kit

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SKU:
HUFI00023
€499

Description

Human VEGFR1 / Flt-1 ELISA Kit - Information

The ELISA Genie VEGFR1 / Flt-1 ELISA Kit can assay for VEGFR1 / Flt-1 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our VEGFR1 / Flt-1 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound VEGFR1 / Flt-1 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human VEGFR1 / Flt-1 ELISA Kit - Data

Description

Tyrosine-protein kinase that acts as a cell-surface receptor for VEGFA, VEGFB and PGF, and plays an essential role in the development of embryonic vasculature, the regulation of angiogenesis, cell survival, cell migration, macrophage function, chemotaxis, and cancer cell invasion. May play an essential role as a negative regulator of embryonic angiogenesis by inhibiting excessive proliferation of endothelial cells. Can promote endothelial cell proliferation, survival and angiogenesis in adulthood. Its function in promoting cell proliferation seems to be cell-type specific. Promotes PGF-mediated proliferation of endothelial cells, proliferation of some types of cancer cells, but does not promote proliferation of normal fibroblasts (in vitro). Has very high affinity for VEGFA and relatively low protein kinase activity; may function as a negative regulator of VEGFA signaling by limiting the amount of free VEGFA and preventing its binding to KDR. Likewise, isoforms lacking a transmembrane domain, such as isoform 2, isoform 3 and isoform 4, may function as decoy receptors for VEGFA. Modulates KDR signaling by forming heterodimers with KDR. Ligand binding leads to the activation of several signaling cascades. Activation of PLCG leads to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate and the activation of protein kinase C. Mediates phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3-kinase, leading to activation of phosphatidylinositol kinase and the downstream signaling pathway. Mediates activation of MAPK1/ERK2, MAPK3/ERK1 and the MAP kinase signaling pathway, as well as of the AKT1 signaling pathway. Phosphorylates SRC and YES1, and may also phosphorylate CBL. Isoform 1 phosphorylates PLCG. Promotes phosphorylation of AKT1 at 'Ser-473'. Promotes phosphorylation of PTK2/FAK1. Isoform 7 has a truncated kinase domain; it increases phosphorylation of SRC at 'Tyr-418' by unknown means and promotes tumor cell invasion.1

Post-Translational Modification

N-glycosylated. Ubiquitinated after VEGFA-mediated autophosphorylation, leading to proteolytic degradation. Autophosphorylated on tyrosine residues upon ligand binding. Autophosphorylation occurs in trans, i.e. one subunit of the dimeric receptor phosphorylates tyrosine residues on the other subunit. Phosphorylation at Tyr-1169 is important for interaction with PLCG. Phosphorylation at Tyr-1213 is important for interaction with PIK3R1, PTPN11, GRB2, and PLCG. Phosphorylation at Tyr-1333 is important for endocytosis and for interaction with CBL, NCK1 and CRK. Is probably dephosphorylated by PTPRB.

Uniprot ID P17948
Alias

VEGFR1/FLT1(Vascular Endothelial Growth Factor Receptor 1)/FLT1/Flt-1/ FLT-1/Fms-like tyrosine kinase 1/fms-related tyrosine kinase 1(vascular endothelial growth factor/vascularpermeability factor receptor)/FRT/Tyrosine-protein kinase FRT/Tyrosine-protein kinase receptor FLT/vascular endothelial growth factor receptor 1/Vascular permeability factor receptor/VEGFR1/VEGFR-1

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of VEGFR1 concentrations in serum plasma and other biological fluids.

Size

96T

Range

125-8000pg/ml

Sensitivity

< 75pg/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of VEGFR1 and the recovery rates were calculated by comparing the measured value to the expected amount of VEGFR1 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 85-104 97
EDTA plasma(n=5) 92-104 99
UFH plasma(n=5) 89-104 95
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of VEGFR1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 85-98% 88-101% 88-104% 88-103%
EDTA plasma(n=5) 86-101% 87-96% 84-100% 83-97%
UFH plasma(n=5) 88-95% 84-100% 84-98% 83-97%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human VEGFR1 / Flt-1 ELISA Kit Protocol

The below protocol is a sample protocol for Human VEGFR1 / Flt-1 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human VEGFR1 / Flt-1 present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

Sandwich ELISA Protocol

Kit Protocol:

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.

Human VEGFR1 / Flt-1 ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Human VEGFR1 / Flt-1 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Human VEGFR1 / Flt-1 ELISA Kit Protein Information

UniProt Protein Function:VEGFR1: a receptor tyrosine kinase of the VEGFR family. Receptor for VEGF, VEGFB and PGF. The VEGF-kinase ligand/receptor signaling system plays a key role in vascular development and regulation of vascular permeability. Two splice variant isoforms have been described. Isoform SFlt1 may have an inhibitory role in angiogenesis.
UniProt Protein Details:

Protein type:Protein kinase, TK; EC 2.7.10.1; Protein kinase, tyrosine (receptor); Kinase, protein; Membrane protein, integral; TK group; VEGFR family

Chromosomal Location of Human Ortholog: 13q12

Cellular Component: extracellular space; focal adhesion; integral to plasma membrane; plasma membrane; receptor complex; endosome

Molecular Function:identical protein binding; vascular endothelial growth factor receptor activity; protein binding; growth factor binding; transmembrane receptor protein tyrosine kinase activity; ATP binding

Biological Process: cell migration; peptidyl-tyrosine phosphorylation; protein amino acid autophosphorylation; positive regulation of phosphoinositide 3-kinase activity; positive regulation of vascular endothelial growth factor receptor signaling pathway; positive regulation of phosphoinositide 3-kinase cascade; patterning of blood vessels; monocyte chemotaxis; positive regulation of MAP kinase activity; positive regulation of angiogenesis; positive regulation of MAPKKK cascade; positive regulation of cell proliferation; blood vessel morphogenesis; embryonic morphogenesis; sprouting angiogenesis; cell differentiation; vascular endothelial growth factor receptor signaling pathway; transmembrane receptor protein tyrosine kinase signaling pathway; positive regulation of cell migration

NCBI Summary:This gene encodes a member of the vascular endothelial growth factor receptor (VEGFR) family. VEGFR family members are receptor tyrosine kinases (RTKs) which contain an extracellular ligand-binding region with seven immunoglobulin (Ig)-like domains, a transmembrane segment, and a tyrosine kinase (TK) domain within the cytoplasmic domain. This protein binds to VEGFR-A, VEGFR-B and placental growth factor and plays an important role in angiogenesis and vasculogenesis. Expression of this receptor is found in vascular endothelial cells, placental trophoblast cells and peripheral blood monocytes. Multiple transcript variants encoding different isoforms have been found for this gene. Isoforms include a full-length transmembrane receptor isoform and shortened, soluble isoforms. The soluble isoforms are associated with the onset of pre-eclampsia.[provided by RefSeq, May 2009]
UniProt Code:P17948
NCBI GenInfo Identifier:143811474
NCBI Gene ID:2321
NCBI Accession:P17948.2
UniProt Secondary Accession:P17948,O60722, A3E342, A3E344, A8KA71, B0LPF1, B2BF46 B2BF47, B2BF48, B3FR89, B5A923, F5H5L6,
UniProt Related Accession:P17948
Molecular Weight:41,175 Da
NCBI Full Name:Vascular endothelial growth factor receptor 1
NCBI Synonym Full Names:fms-related tyrosine kinase 1
NCBI Official Symbol:FLT1  
NCBI Official Synonym Symbols:FLT; FLT-1; VEGFR1; VEGFR-1  
NCBI Protein Information:vascular endothelial growth factor receptor 1; fms-like tyrosine kinase 1; tyrosine-protein kinase FRT; tyrosine-protein kinase receptor FLT; vascular permeability factor receptor; fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)
UniProt Protein Name:Vascular endothelial growth factor receptor 1
UniProt Synonym Protein Names:Fms-like tyrosine kinase 1; FLT-1; Tyrosine-protein kinase FRT; Tyrosine-protein kinase receptor FLT; FLT; Vascular permeability factor receptor
Protein Family:Vascular endothelial growth factor receptor
UniProt Gene Name:FLT1  
UniProt Entry Name:VGFR1_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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