Human CENPE (Centromere Protein E) ELISA Kit (HUES01839)

SKU:
HUES01839
Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
€539

Description

ELISA Kit Technical ManualMSDS

Human CENPE (Centromere Protein E) 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 CENPE . 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 CENPE 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 CENPE, 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 CENPE. The concentration of Human CENPE 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 CENPE in samples. No significant cross-reactivity or interference between Human CENPE 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
202.339
2.397
2.3682.304
101.69
1.702
1.6961.632
50.904
0.896
0.90.836
2.50.433
0.437
0.4350.371
1.250.259
0.247
0.2530.189
0.630.174
0.16
0.1670.103
0.310.111
0.121
0.1160.052
00.058
0.07
0.064--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)1.112.249.131.152.039.19
Standard deviation0.070.120.470.060.100.48
C V (%)6.315.365.155.224.935.22

Recovery

The recovery of Human CENPE 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)83-9790
EDTA plasma (n=5)87-9993
Cell culture media (n=5)86-9791

Linearity

Samples were spiked with high concentrations of Human CENPE 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 (%)86-9985-9699-116
Average (%)9391106
1:4Range (%)89-10382-9487-99
Average (%)958994
1:8Range (%)87-10286-10085-99
Average (%)939291
1:16Range (%)89-10285-10185-98
Average (%)969292

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 CENPE (Centromere Protein E) ELISA Kit (HUES01839) 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 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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 CENPE (Centromere Protein E) ELISA Kit (HUES01839) Protein Information

UniProt Protein Function:CENPE: Essential for the maintenance of chromosomal stability through efficient stabilization of microtubule capture at kinetochores. Plays a key role in the movement of chromosomes toward the metaphase plate during mitosis. Is a slow plus end- directed motor whose activity is essential for metaphase chromosome alignment. Couples chromosome position to microtubule depolymerizing activity. The highly processive microtubule- dependent motor activity of CENPE serves to power chromosome congression and provides a flexible, motile tether linking kinetochores to dynamic spindle microtubules. Necessary for the mitotic checkpoint signal at individual kinetochores to prevent aneuploidy due to single chromosome loss. Required for the efficient recruitment of BUBR1, MAD1 and MAD2 to attached and newly unattached kinetochores. Stimulates mammalian BUBR1 kinase activity. Accumulates just before mitosis at the G2 phase of the cell cycle. Belongs to the kinesin-like protein family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Microtubule-binding; Motor

Chromosomal Location of Human Ortholog: 4q24

Cellular Component: chromosome; chromosome, centromeric region; cytosol; kinesin complex; kinetochore; kinetochore microtubule; membrane; microtubule; microtubule cytoskeleton; midbody; nucleus; outer kinetochore of condensed chromosome

Molecular Function:ATP binding; ATPase activity; kinetochore binding; microtubule binding; microtubule motor activity; protein binding

Biological Process: antigen processing and presentation of exogenous peptide antigen via MHC class II; attachment of mitotic spindle microtubules to kinetochore; cell division; chromosome segregation; kinetochore assembly; metaphase plate congression; microtubule-based movement; mitotic cell cycle; mitotic chromosome movement towards spindle pole; mitotic metaphase plate congression; mitotic spindle organization and biogenesis; multicellular organism development; positive regulation of protein kinase activity; regulation of mitotic metaphase/anaphase transition; retrograde vesicle-mediated transport, Golgi to ER; sister chromatid cohesion

Disease: Microcephaly 13, Primary, Autosomal Recessive

NCBI Summary:Centrosome-associated protein E (CENPE) is a kinesin-like motor protein that accumulates in the G2 phase of the cell cycle. Unlike other centrosome-associated proteins, it is not present during interphase and first appears at the centromere region of chromosomes during prometaphase. This protein is required for stable spindle microtubule capture at kinetochores which is a necessary step in chromosome alignment during prometaphase. This protein also couples chromosome position to microtubule depolymerizing activity. Alternative splicing results in multiple transcript variants encoding distinct protein isoforms. [provided by RefSeq, Nov 2014]
UniProt Code:Q02224
NCBI GenInfo Identifier:160358869
NCBI Gene ID:1062
NCBI Accession:Q02224.2
UniProt Secondary Accession:Q02224,Q4LE75, A6NKY9, A8K2U7,
UniProt Related Accession:Q02224
Molecular Weight:301,789 Da
NCBI Full Name:Centromere-associated protein E
NCBI Synonym Full Names:centromere protein E
NCBI Official Symbol:CENPE
NCBI Official Synonym Symbols:KIF10; CENP-E; MCPH13; PPP1R61
NCBI Protein Information:centromere-associated protein E
UniProt Protein Name:Centromere-associated protein E
UniProt Synonym Protein Names:Centromere protein E; CENP-E; Kinesin-7
Protein Family:Centromere-associated protein
UniProt Gene Name:CENPE
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