Human NEK2(Serine/threonine-protein kinase Nek2) ELISA Kit

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SKU:
HUFI03063
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P51955
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich ELISA, Double Antigen
Synonyms:
HSPK 21, Never in mitosis A-related kinase 2, NimA-related protein kinase 2, NimA-like protein kinase 1, NEK2A, NLK1
Reactivity:
Human
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Description

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Product Name:Human NEK2(Serine/threonine-protein kinase Nek2) ELISA Kit
Product Code:HUFI03063
Size:96 Assays
Alias:HSPK 21, Never in mitosis A-related kinase 2, NimA-related protein kinase 2, NimA-like protein kinase 1, NEK2A, NLK1
Detection method:Sandwich ELISA, Double Antigen
Application:This immunoassay kit allows for the in vitro quantitative determination of Human NEK2 concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human NEK2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human NEK2 in samples. Enquire for more information.
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NEK2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
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 Antigen (Concentrated)120ul4°C (Protect from light)
Antigen 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
UniprotP51955
UniProt Protein Function:NEK2: a protein kinase of the NEK family. Accumulates throughout S phase and shows maximal levels in late G2. This expression pattern is highly reminiscent of that of A and B cyclins. Phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation
UniProt Protein Details:Protein type:Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Nucleolus; Protein kinase, Other; EC 2.7.11.1; Other group; NEK family

Chromosomal Location of Human Ortholog: 1q32.3

Cellular Component: kinetochore; spindle pole; microtubule; centrosome; intercellular bridge; protein complex; condensed nuclear chromosome; cytoplasm; nucleolus; midbody; nucleus; cytosol

Molecular Function:protein serine/threonine kinase activity; protein binding; metal ion binding; protein phosphatase binding; ATP binding; protein kinase activity

Biological Process: mitosis; blastocyst development; regulation of attachment of spindle microtubules to kinetochore; mitotic sister chromatid segregation; protein amino acid autophosphorylation; regulation of mitotic centrosome separation; organelle organization and biogenesis; centrosome separation; spindle assembly; protein amino acid phosphorylation; negative regulation of DNA binding; chromosome segregation; meiotic cell cycle; cell division; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; regulation of mitosis; mitotic cell cycle; G2/M transition of mitotic cell cycle

Disease: Retinitis Pigmentosa 67

NCBI Summary:This gene encodes a serine/threonine-protein kinase that is involved in mitotic regulation. This protein is localized to the centrosome, and undetectable during G1 phase, but accumulates progressively throughout the S phase, reaching maximal levels in late G2 phase. Alternatively spliced transcript variants encoding different isoforms with distinct C-termini have been noted for this gene. [provided by RefSeq, Feb 2011]
UniProt Code:P51955
NCBI GenInfo Identifier:1709252
NCBI Gene ID:4751
NCBI Accession:P51955.1
UniProt Secondary Accession:P51955,Q53FD6, Q5I1Z9, Q5VXZ1, Q6NZX8, Q7Z634, Q86XH2 Q96QN9,
UniProt Related Accession:P51955
Molecular Weight:445
NCBI Full Name:Serine/threonine-protein kinase Nek2
NCBI Synonym Full Names:NIMA-related kinase 2
NCBI Official Symbol:NEK2
NCBI Official Synonym Symbols:NLK1; RP67; NEK2A; HsPK21; PPP1R111
NCBI Protein Information:serine/threonine-protein kinase Nek2; nimA-like protein kinase 1; nimA-related protein kinase 2; protein phosphatase 1, regulatory subunit 111; NIMA (never in mitosis gene a)-related kinase 2
UniProt Protein Name:Serine/threonine-protein kinase Nek2
UniProt Synonym Protein Names:HSPK 21; Never in mitosis A-related kinase 2; NimA-related protein kinase 2; NimA-like protein kinase 1
Protein Family:Serine/threonine-protein kinase
UniProt Gene Name:NEK2
UniProt Entry Name:NEK2_HUMAN

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

Bring all reagents and samples to room temperature 30 minutes before use. 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 (diluted at least ½ with Sample Dilution Buffer) and control (zero) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells.
2.Aliquot 100µl of standard solutions into the standard wells
3.Add 100µl of Sample / Standard dilution buffer into the control (blank) well.
4.Add 100µl of properly diluted sample (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 antigen 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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