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Human Netrin-1 ELISA Kit

SKU:
HUFI01636
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O95631
Sensitivity:
18.75pg/ml
Range:
31.25-2000pg/ml
ELISA Type:
Sandwich
Synonyms:
Ntn1, Netrin 1, netrin-1, Lnetrin 1, Mouse, homolog of
Reactivity:
Human
Research Area:
Cell Death
€599
Frequently bought together:

Description

Human Netrin-1 ELISA

Netrin belongs to a family of secreted laminin-related proteins. Netrin's purpose is unknown, although it is believed to play a role in axon guidance and cell migration during development. Netrin abnormalities and loss of expression suggest that netrin variation might be linked to cancer development. Cell junction organization and activation of cAMP-dependent PKA are two pathways related to Netrin. The Assay Genie Human Netrin-1 ELISA Kit is a highly sensitive assay for the quantitative measurement of Netrin-1 in serum, blood, plasma, cell culture supernatant and tissue samples.

system_update_altDatasheetsystem_update_altMSDS
Product Name:Human Netrin-1 ELISA Kit
Product Code:HUFI01636
Size:96 Assays
Alias:Ntn1, Netrin 1, netrin-1, Lnetrin 1, Mouse, homolog of
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human NTN1 concentrations in serum plasma and other biological fluids.
Sensitivity:18.75pg/ml
Range:31.25-2000pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human NTN1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human NTN1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)86-9994
EDTA plasma(n=5)86-10595
UFH plasma(n=5)94-103100
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NTN1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)86-103%92-105%85-103%
EDTA plasma(n=5)83-100%82-99%88-101%
UFH plasma(n=5)83-99%82-97%80-96%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
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:

  • 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
UniprotO95631
UniProt Protein Function:NTN1: Netrins control guidance of CNS commissural axons and peripheral motor axons. Its association with either DCC or some UNC5 receptors will lead to axon attraction or repulsion, respectively. It also serve as a survival factor via its association with its receptors which prevent the initiation of apoptosis. Involved in tumorigenesis by regulating apoptosis.
UniProt Protein Details:Protein type:Secreted; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 17p13.1

Cellular Component: extracellular region

Molecular Function:protein binding

Biological Process: axon guidance; Cdc42 protein signal transduction; positive regulation of axon extension; Ras protein signal transduction; substrate-bound cell migration, cell extension

NCBI Summary:Netrin is included in a family of laminin-related secreted proteins. The function of this gene has not yet been defined; however, netrin is thought to be involved in axon guidance and cell migration during development. Mutations and loss of expression of netrin suggest that variation in netrin may be involved in cancer development. [provided by RefSeq, Jul 2008]
UniProt Code:O95631
NCBI GenInfo Identifier:229462906
NCBI Gene ID:9423
NCBI Accession:O95631.2
UniProt Secondary Accession:O95631,E9KL51,
UniProt Related Accession:O95631
Molecular Weight:67,748 Da
NCBI Full Name:Netrin-1
NCBI Synonym Full Names:netrin 1
NCBI Official Symbol:NTN1  
NCBI Official Synonym Symbols:NTN1L  
NCBI Protein Information:netrin-1
UniProt Protein Name:Netrin-1
UniProt Synonym Protein Names:Epididymis tissue protein Li 131P
Protein Family:Netrin
UniProt Gene Name:NTN1  
UniProt Entry Name:NET1_HUMAN

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

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.

StepProtocol
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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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.

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