Human NUP62 (Nucleoporin 62kDa) ELISA Kit (HUES03267)

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HUES03267
€539

Description

ELISA Kit Technical ManualMSDS

Human NUP62 (Nucleoporin 62kDa) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human NUP62 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human NUP62 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human NUP62, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human NUP62. The concentration of Human NUP62 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human NUP62 in samples. No significant cross-reactivity or interference between Human NUP62 and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(ng/mL)
O.D Average Corrected
202.273
2.315
2.2942.212
101.514
1.516
1.5151.433
50.853
0.827
0.840.758
2.50.465
0.491
0.4780.396
1.250.266
0.25
0.2580.176
0.630.19
0.162
0.1760.094
0.310.125
0.135
0.130.048
00.08
0.084
0.082--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NUP62 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NUP62 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.952.318.741.022.518.58
Standard deviation0.070.130.270.070.150.30
C V (%)7.375.633.096.865.983.50

Recovery

The recovery of Human NUP62 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)91-10397
EDTA plasma (n=5)86-9792
Cell culture media (n=5)91-10696

Linearity

Samples were spiked with high concentrations of Human NUP62 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)90-103100-11287-101
Average (%)9510695
1:4Range (%)87-10183-9884-98
Average (%)948991
1:8Range (%)93-10587-9785-96
Average (%)999291
1:16Range (%)87-9886-9883-96
Average (%)929190

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human NUP62 (Nucleoporin 62kDa) ELISA Kit (HUES03267) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human NUP62 (Nucleoporin 62kDa) ELISA Kit (HUES03267) Protein Information

UniProt Protein Function:NUP62: Essential component of the nuclear pore complex. The N- terminal is probably involved in nucleocytoplasmic transport. The C-terminal is probably involved in protein-protein interaction via coiled-coil formation and may function in anchorage of p62 to the pore complex. Defects in NUP62 are the cause of infantile striatonigral degeneration (SNDI); also known as infantile bilateral striatal necrosis (IBSN) or familial striatal degeneration. SNDI is a neurological disorder characterized by symmetrical degeneration of the caudate nucleus, putamen and occasionally the globus pallidus, with little involvement of the rest of the brain. The clinical features include developmental regression, choreoathetosis, dystonia, spasticity, dysphagia, failure to thrive, nystagmus, optic atrophy and mental retardation. Belongs to the nucleoporin NSP1/NUP62 family.
UniProt Protein Details:

Protein type:Nucleoporin; Adaptor/scaffold

Chromosomal Location of Human Ortholog: 19q13.33

Cellular Component: pore complex; spindle pole; nucleocytoplasmic shuttling complex; nuclear membrane; intracellular membrane-bound organelle; cytoplasm; nuclear envelope; nuclear pore; ribonucleoprotein complex

Molecular Function:ubiquitin binding; protein binding; receptor signaling complex scaffold activity; structural constituent of nuclear pore; PTB domain binding; chromatin binding; SH2 domain binding; thyroid hormone receptor binding; nucleocytoplasmic transporter activity

Biological Process: cell death; negative regulation of epidermal growth factor receptor signaling pathway; negative regulation of MAP kinase activity; mRNA transport; viral reproduction; hormone-mediated signaling; positive regulation of transcription, DNA-dependent; mitotic nuclear envelope disassembly; regulation of signal transduction; nucleocytoplasmic transport; pathogenesis; viral infectious cycle; glucose transport; negative regulation of cell proliferation; protein transport; cell surface receptor linked signal transduction; positive regulation of epidermal growth factor receptor signaling pathway; negative regulation of programmed cell death; negative regulation of Ras protein signal transduction; viral transcription; transmembrane transport; regulation of Ras protein signal transduction; positive regulation of I-kappaB kinase/NF-kappaB cascade; transcription, DNA-dependent; cytokine and chemokine mediated signaling pathway; cell aging; hexose transport; carbohydrate metabolic process; gene expression; mitotic cell cycle; negative regulation of apoptosis

Disease: Striatonigral Degeneration, Infantile

NCBI Summary:The nuclear pore complex is a massive structure that extends across the nuclear envelope, forming a gateway that regulates the flow of macromolecules between the nucleus and the cytoplasm. Nucleoporins are the main components of the nuclear pore complex in eukaryotic cells. The protein encoded by this gene is a member of the FG-repeat containing nucleoporins and is localized to the nuclear pore central plug. This protein associates with the importin alpha/beta complex which is involved in the import of proteins containing nuclear localization signals. Multiple transcript variants of this gene encode a single protein isoform. [provided by RefSeq, Jul 2008]
UniProt Code:P37198
NCBI GenInfo Identifier:134047855
NCBI Gene ID:23636
NCBI Accession:P37198.3
UniProt Related Accession:P37198
Molecular Weight:
NCBI Full Name:Nuclear pore glycoprotein p62
NCBI Synonym Full Names:nucleoporin 62
NCBI Official Symbol:NUP62  
NCBI Official Synonym Symbols:p62; IBSN; SNDI  
NCBI Protein Information:nuclear pore glycoprotein p62
UniProt Protein Name:Nuclear pore glycoprotein p62
UniProt Synonym Protein Names:62 kDa nucleoporin; Nucleoporin Nup62
Protein Family:Nuclear pore glycoprotein
UniProt Gene Name:NUP62  
UniProt Entry Name:NUP62_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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