Human CCNB (Cyclin-B) ELISA Kit (HUES01523)

SKU:
HUES01523
€539

Description

ELISA Kit Technical ManualMSDS

Human CCNB (Cyclin-B) 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 CCNB . 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 CCNB 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 CCNB, 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 CCNB. The concentration of Human CCNB 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 CCNB in samples. No significant cross-reactivity or interference between Human CCNB 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
202.457
2.499
2.4782.393
101.678
1.68
1.6791.594
50.927
0.903
0.9150.83
2.50.437
0.447
0.4420.357
1.250.256
0.25
0.2530.168
0.630.189
0.181
0.1850.1
0.310.132
0.14
0.1360.051
00.075
0.095
0.085--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.942.898.630.992.838.10
Standard deviation0.060.160.310.070.160.36
C V (%)6.385.543.597.075.654.44

Recovery

The recovery of Human CCNB 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)92-10398
EDTA plasma (n=5)88-10495
Cell culture media (n=5)95-106100

Linearity

Samples were spiked with high concentrations of Human CCNB 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 (%)88-10295-10892-106
Average (%)9310198
1:4Range (%)92-10580-9482-95
Average (%)1008787
1:8Range (%)90-10679-9083-96
Average (%)978588
1:16Range (%)89-10180-9388-98
Average (%)968693

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 CCNB (Cyclin-B) ELISA Kit (HUES01523) 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 CCNB (Cyclin-B) ELISA Kit (HUES01523) Protein Information

UniProt Protein Function:CCNB1: a member of the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. Complexes with cdc2 to form the maturation-promoting factor (MPF). Two alternatively spliced isoforms have been found, a constitutively expressed form and a cell cycle-regulated form that is expressed predominantly during G2/M phase.
UniProt Protein Details:

Protein type:Cell cycle regulation; Activator

Chromosomal Location of Human Ortholog: 5q12

Cellular Component: nucleoplasm; spindle pole; centrosome; membrane; cytoplasm; nucleus; cytosol

Molecular Function:protein binding; patched binding; histone kinase activity; protein kinase binding

Biological Process: positive regulation of ubiquitin-protein ligase activity during mitotic cell cycle; positive regulation of histone phosphorylation; regulation of cell cycle; ventricular cardiac muscle cell development; positive regulation of attachment of spindle microtubules to kinetochore; mitotic nuclear envelope disassembly; positive regulation of mRNA 3'-end processing; positive regulation of mitotic cell cycle; positive regulation of fibroblast proliferation; negative regulation of protein amino acid phosphorylation; tissue regeneration; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; protein complex assembly; G2/M transition of mitotic cell cycle; positive regulation of cardiac muscle cell proliferation; response to drug; in utero embryonic development; oocyte maturation; mitotic metaphase plate congression; response to DDT; gut development; regulation of ubiquitin-protein ligase activity during mitotic cell cycle; response to mechanical stimulus; cell division; spermatogenesis; regulation of cyclin-dependent protein kinase activity; mitotic cell cycle; mitotic spindle stabilization; G1/S transition of mitotic cell cycle

NCBI Summary:The protein encoded by this gene is a regulatory protein involved in mitosis. The gene product complexes with p34(cdc2) to form the maturation-promoting factor (MPF). Two alternative transcripts have been found, a constitutively expressed transcript and a cell cycle-regulated transcript, that is expressed predominantly during G2/M phase. The different transcripts result from the use of alternate transcription initiation sites. [provided by RefSeq, Jul 2008]
UniProt Code:P14635
NCBI GenInfo Identifier:116176
NCBI Gene ID:891
NCBI Accession:P14635.1
UniProt Secondary Accession:P14635,Q5TZP9, A8K066,
UniProt Related Accession:P14635
Molecular Weight:44,102 Da
NCBI Full Name:G2/mitotic-specific cyclin-B1
NCBI Synonym Full Names:cyclin B1
NCBI Official Symbol:CCNB1
NCBI Official Synonym Symbols:CCNB
NCBI Protein Information:G2/mitotic-specific cyclin-B1; G2/mitotic-specific cyclin B1
UniProt Protein Name:G2/mitotic-specific cyclin-B1
UniProt Gene Name:CCNB1
UniProt Entry Name:CCNB1_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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