Human NUP98 (Nucleoporin 98kDa) ELISA Kit (HUES02420)

SKU:
HUES02420
Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
€539

Description

ELISA Kit Technical ManualMSDS

Human NUP98 (Nucleoporin 98kDa) 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 NUP98 . 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 NUP98 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 NUP98, 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 NUP98. The concentration of Human NUP98 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 NUP98 in samples. No significant cross-reactivity or interference between Human NUP98 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
202.374
2.428
2.4012.309
101.623
1.665
1.6441.552
50.946
0.91
0.9280.836
2.50.462
0.482
0.4720.38
1.250.267
0.253
0.260.168
0.630.192
0.19
0.1910.099
0.310.137
0.147
0.1420.05
00.088
0.096
0.092--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NUP98 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NUP98 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.991.959.961.031.9910.78
Standard deviation0.070.110.510.050.120.58
C V (%)7.075.645.124.856.035.38

Recovery

The recovery of Human NUP98 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)91-10398
EDTA plasma (n=5)90-10697
Cell culture media (n=5)86-9993

Linearity

Samples were spiked with high concentrations of Human NUP98 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 (%)99-11494-11096-109
Average (%)105101103
1:4Range (%)90-10580-9287-99
Average (%)978593
1:8Range (%)93-10880-9581-93
Average (%)1008787
1:16Range (%)93-10482-9688-99
Average (%)988893

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 NUP98 (Nucleoporin 98kDa) ELISA Kit (HUES02420) 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 NUP98 (Nucleoporin 98kDa) ELISA Kit (HUES02420) Protein Information

UniProt Protein Function:NUP98: Nup98 and Nup96 play a role in the bidirectional transport across the nucleoporin complex (NPC). The repeat domain in Nup98 has a direct role in the transport. Nup98 interacts directly with Nup96. Nup96 is part of the Nup160 subcomplex in the nuclear pore which is composed of Nup160, Nup133, Nup107 and Nup96. This complex plays a role in RNA export and in tethering Nup98 and Nup153 to the nucleus. May interact with RAE1. Interacts with vesicular stomatitis virus protein M. Belongs to the nucleoporin GLFG family. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Nucleoporin

Chromosomal Location of Human Ortholog: 11p15.5

Cellular Component: nucleoplasm; kinetochore; nuclear membrane; nuclear pore; nuclear envelope; cytosol; nuclear inclusion body

Molecular Function:protein binding; structural constituent of nuclear pore; transporter activity; peptide binding

Biological Process: nuclear pore complex assembly; mRNA transport; viral reproduction; protein import into nucleus, docking; cytokine and chemokine mediated signaling pathway; mitotic nuclear envelope disassembly; nucleocytoplasmic transport; pathogenesis; viral infectious cycle; glucose transport; nuclear pore organization and biogenesis; hexose transport; carbohydrate metabolic process; viral transcription; gene expression; mitotic cell cycle; DNA replication; transmembrane transport

NCBI Summary:Signal-mediated nuclear import and export proceed through the nuclear pore complex (NPC), which is comprised of approximately 50 unique proteins collectively known as nucleoporins. The 98 kDa nucleoporin is generated through a biogenesis pathway that involves synthesis and proteolytic cleavage of a 186 kDa precursor protein. This cleavage results in the 98 kDa nucleoporin as well as a 96 kDa nucleoporin, both of which are localized to the nucleoplasmic side of the NPC. Rat studies show that the 98 kDa nucleoporin functions as one of several docking site nucleoporins of transport substrates. The human gene has been shown to fuse to several genes following chromosome translocations in acute myelogenous leukemia (AML) and T-cell acute lymphocytic leukemia (T-ALL). This gene is one of several genes located in the imprinted gene domain of 11p15.5, an important tumor-suppressor gene region. Alterations in this region have been associated with the Beckwith-Wiedemann syndrome, Wilms tumor, rhabdomyosarcoma, adrenocortical carcinoma, and lung, ovarian, and breast cancer. Alternative splicing of this gene results in several transcript variants; however, not all variants have been fully described. [provided by RefSeq, May 2010]
UniProt Code:P52948
NCBI GenInfo Identifier:308153660
NCBI Gene ID:4928
NCBI Accession:P52948.4
UniProt Secondary Accession:P52948,Q8IUT2, Q8WYB0, Q96E54, Q9H3Q4, Q9NT02, Q9UF57 Q9UHX0, Q9Y6J4, Q9Y6J5,
UniProt Related Accession:P52948
Molecular Weight:1817
NCBI Full Name:Nuclear pore complex protein Nup98-Nup96
NCBI Synonym Full Names:nucleoporin 98kDa
NCBI Official Symbol:NUP98
NCBI Official Synonym Symbols:ADIR2; NUP96; NUP196
NCBI Protein Information:nuclear pore complex protein Nup98-Nup96; nucleoporin 98kD; GLFG-repeat containing nucleoporin
UniProt Protein Name:Nuclear pore complex protein Nup98-Nup96
UniProt Synonym Protein Names:98 kDa nucleoporin; Nucleoporin Nup98; Nup98
UniProt Gene Name:NUP98
UniProt Entry Name:NUP98_HUMAN
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