Human Neuropilin 1 ELISA Kit

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SKU:
HUFI01644
€299

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human Neuropilin 1 ELISA Kit - Information

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

How our Neuropilin 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 Neuropilin 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 Neuropilin 1 ELISA Kit - Data

Description

The membrane-bound isoform 1 is a receptor involved in the development of the cardiovascular system, in angiogenesis, in the formation of certain neuronal circuits and in organogenesis outside the nervous system. It mediates the chemorepulsant activity of semaphorins. It binds to semaphorin 3A, The PLGF-2 isoform of PGF, The VEGF165 isoform of VEGFA and VEGFB. Coexpression with KDR results in increased VEGF165 binding to KDR as well as increased chemotaxis. Regulate VEGF-induced angiogenesis. Binding to VEGFA initiates a signaling pathway needed for motor neuron axon guidance and cell body migration, including for the caudal migration of facial motor neurons from rhombomere 4 to rhombomere 6 during embryonic development.; The soluble isoform 2 binds VEGF-165 and appears to inhibit its binding to cells. It may also induce apoptosis by sequestering VEGF-165. May bind as well various members of the semaphorin family. Its expression has an averse effect on blood vessel number and integrity.

Post-Translational Modification

Uniprot ID O14786
Alias

NRP1(Neuropilin 1)/BDCA-4/CD304/BDCA4/CD304 antigen/neuropilin 1/neuropilin-1/NRPNP1/transmembrane receptor/Vascular endothelial cell growth factor 165 receptor/VEGF165R

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of NRP1 concentrations in serum plasma and other biological fluids.

Size

96T

Range

78.125-5000pg/ml

Sensitivity

< 46.875pg/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of NRP1 and the recovery rates were calculated by comparing the measured value to the expected amount of NRP1 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 86-103 95
EDTA plasma(n=5) 93-105 99
UFH plasma(n=5) 86-103 98
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of NRP1 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) 87-105% 87-102% 86-100% 88-104%
EDTA plasma(n=5) 89-97% 90-99% 82-93% 86-101%
UFH plasma(n=5) 83-93% 85-93% 82-100% 90-100%
CV(%)

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

Note

For Research Use Only

Human Neuropilin 1 ELISA Kit Protocol

The below protocol is a sample protocol for Human Neuropilin 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 Neuropilin 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 Neuropilin 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 Neuropilin 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 Neuropilin 1 ELISA Kit Protein Information

UniProt Protein Function:NRP1: The membrane-bound isoform 1 is a receptor involved in the development of the cardiovascular system, in angiogenesis, in the formation of certain neuronal circuits and in organogenesis outside the nervous system. It mediates the chemorepulsant activity of semaphorins. It binds to semaphorin 3A, The PLGF-2 isoform of PGF, The VEGF-165 isoform of VEGF and VEGF-B. Coexpression with KDR results in increased VEGF-165 binding to KDR as well as increased chemotaxis. It may regulate VEGF-induced angiogenesis. Homodimer, and heterodimer with NRP2. Interacts with FER. Binds PLXNB1. The expression of isoforms 1 and 2 does not seem to overlap. Isoform 1 is expressed by the blood vessels of different tissues. In the developing embryo it is found predominantly in the nervous system. In adult tissues, it is highly expressed in heart and placenta; moderately in lung, liver, skeletal muscle, kidney and pancreas; and low in adult brain. Isoform 2 is found in liver hepatocytes, kidney distal and proximal tubules. Belongs to the neuropilin family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Receptor, misc.

Chromosomal Location of Human Ortholog: 10p12

Cellular Component: axon; cytoplasmic vesicle; cytosol; early endosome; extracellular space; focal adhesion; plasma membrane; receptor complex

Molecular Function:coreceptor activity; growth factor binding; protein binding; vascular endothelial growth factor receptor activity

Biological Process: angiogenesis; artery morphogenesis; axon extension involved in axon guidance; cell migration during sprouting angiogenesis; cell-cell signaling; hepatocyte growth factor receptor signaling pathway; nerve development; neuron migration; organ morphogenesis; patterning of blood vessels; platelet-derived growth factor receptor signaling pathway; positive chemotaxis; positive regulation of axon extension involved in axon guidance; positive regulation of endothelial cell proliferation; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of smooth muscle cell migration; retinal ganglion cell axon guidance; signal transduction; sprouting angiogenesis; vascular endothelial growth factor receptor signaling pathway

NCBI Summary:This gene encodes one of two neuropilins, which contain specific protein domains which allow them to participate in several different types of signaling pathways that control cell migration. Neuropilins contain a large N-terminal extracellular domain, made up of complement-binding, coagulation factor V/VIII, and meprin domains. These proteins also contains a short membrane-spanning domain and a small cytoplasmic domain. Neuropilins bind many ligands and various types of co-receptors; they affect cell survival, migration, and attraction. Some of the ligands and co-receptors bound by neuropilins are vascular endothelial growth factor (VEGF) and semaphorin family members. Several alternatively spliced transcript variants that encode different protein isoforms have been described for this gene. [provided by RefSeq, Oct 2011]
UniProt Code:O14786
NCBI GenInfo Identifier:206729912
NCBI Gene ID:8829
NCBI Accession:O14786.3
UniProt Secondary Accession:O14786,O60461, Q5T7F1, Q5T7F2, Q5T7F3, Q86T59, Q96I90 Q96IH5, B0LPG9,
UniProt Related Accession:O14786
Molecular Weight:68,376 Da
NCBI Full Name:Neuropilin-1
NCBI Synonym Full Names:neuropilin 1
NCBI Official Symbol:NRP1  
NCBI Official Synonym Symbols:NP1; NRP; BDCA4; CD304; VEGF165R  
NCBI Protein Information:neuropilin-1
UniProt Protein Name:Neuropilin-1
UniProt Synonym Protein Names:Vascular endothelial cell growth factor 165 receptor; CD_antigen: CD304
Protein Family:Neuropilin
UniProt Gene Name:NRP1  
UniProt Entry Name:NRP1_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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