Human KPNA2 / Karyopherin alpha 2 ELISA Kit

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SKU:
HUFI01029
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P52292
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
KPNA2, Importin subunit alpha-2, Importin alpha 1, IPOA1, KPNA2, Pendulin, QIP2, RCH1, SRP1-alpha, importin-alpha-P1, karyopherin alpha 2, RAG cohort 1, importin alpha 1, Karyopherin subunit alpha-2, importin alpha 2, RAG cohort 1, RAG cohort protein
Reactivity:
Human
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Description

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Human KPNA2 / Karyopherin alpha 2 ELISA Kit

V(D)J recombination is a process in which parts of two chromosomes combine to form new genetic information. KPNA2 is thought to be involved in V(D)J recombination. KPNA2 is associated with a number of illnesses, including Venezuelan equine encephalitis and influenza.The Assay Genie KPNA2/Karyopherin alpha 2 ELISA is a highly sensitive assay for the quantitative measurement of KPNA2/Karyopherin alpha 2 in serum, blood, plasma, cell culture supernatant and tissue samples.

Product Name:Human KPNA2 / Karyopherin alpha 2 ELISA Kit
Product Code:HUFI01029
Size:96 Assays
Alias:KPNA2, Importin subunit alpha-2, Importin alpha 1, IPOA1, KPNA2, Pendulin, QIP2, RCH1, SRP1-alpha, importin-alpha-P1, karyopherin alpha 2, RAG cohort 1, importin alpha 1, Karyopherin subunit alpha-2, importin alpha 2, RAG cohort 1, RAG cohort protein 1, importin subunit alpha-2, SRP1, SRP1alpha
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human KPNA2 concentrations in serum plasma and other biological fluids.
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human KPNA2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human KPNA2 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)90-10396
EDTA plasma(n=5)86-10191
UFH plasma(n=5)87-9591
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human KPNA2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-103%88-101%85-103%
EDTA plasma(n=5)85-97%86-101%82-91%
UFH plasma(n=5)80-99%81-99%80-99%
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
UniprotP52292
UniProt Protein Function:KPNA2: Functions in nuclear protein import as an adapter protein for nuclear receptor KPNB1. Binds specifically and directly to substrates containing either a simple or bipartite NLS motif. Docking of the importin/substrate complex to the nuclear pore complex (NPC) is mediated by KPNB1 through binding to nucleoporin FxFG repeats and the complex is subsequently translocated through the pore by an energy requiring, Ran- dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds to importin-beta and the three components separate and importin-alpha and -beta are re-exported from the nucleus to the cytoplasm where GTP hydrolysis releases Ran from importin. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus. Belongs to the importin alpha family.
UniProt Protein Details:Protein type:Nuclear import; Karyopherin; Nucleoporin

Chromosomal Location of Human Ortholog: 17q24.2

Cellular Component: nucleoplasm; membrane; cytoplasm; cytosol

Molecular Function:protein binding; histone deacetylase binding; nuclear localization sequence binding; protein transporter activity

Biological Process: viral reproduction; cytokine and chemokine mediated signaling pathway; regulation of DNA recombination; NLS-bearing substrate import into nucleus; DNA metabolic process

NCBI Summary:The import of proteins into the nucleus is a process that involves at least 2 steps. The first is an energy-independent docking of the protein to the nuclear envelope and the second is an energy-dependent translocation through the nuclear pore complex. Imported proteins require a nuclear localization sequence (NLS) which generally consists of a short region of basic amino acids or 2 such regions spaced about 10 amino acids apart. Proteins involved in the first step of nuclear import have been identified in different systems. These include the Xenopus protein importin and its yeast homolog, SRP1 (a suppressor of certain temperature-sensitive mutations of RNA polymerase I in Saccharomyces cerevisiae), which bind to the NLS. KPNA2 protein interacts with the NLSs of DNA helicase Q1 and SV40 T antigen and may be involved in the nuclear transport of proteins. KPNA2 also may play a role in V(D)J recombination [provided by RefSeq, Jul 2008]
UniProt Code:P52292
NCBI GenInfo Identifier:1708480
NCBI Gene ID:3838
NCBI Accession:P52292.1
UniProt Secondary Accession:P52292,Q53YE3, Q9BRU5, B9EJD6,
UniProt Related Accession:P52292
Molecular Weight:529
NCBI Full Name:Importin subunit alpha-1
NCBI Synonym Full Names:karyopherin alpha 2 (RAG cohort 1, importin alpha 1)
NCBI Official Symbol:KPNA2
NCBI Official Synonym Symbols:QIP2; RCH1; IPOA1; SRP1alpha
NCBI Protein Information:importin subunit alpha-1; pendulin; SRP1-alpha; RAG cohort 1; importin alpha 1; importin alpha 2; importin-alpha-P1; RAG cohort protein 1; importin subunit alpha-2; karyopherin subunit alpha-2
UniProt Protein Name:Importin subunit alpha-1
UniProt Synonym Protein Names:Karyopherin subunit alpha-2; RAG cohort protein 1; SRP1-alpha
UniProt Gene Name:KPNA2
UniProt Entry Name:IMA1_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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