Mouse CCNE2 (Cyclin-E2) ELISA Kit (MOES01652)

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MOES01652
€549

Description

ELISA Kit Technical ManualMSDS

Mouse CCNE2 (Cyclin-E2) 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 Mouse CCNE2 . 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 Mouse CCNE2 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 Mouse CCNE2, 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 Mouse CCNE2. The concentration of Mouse CCNE2 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection MethodColormetric
Detection Range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Mouse CCNE2 in samples. No significant cross-reactivity or interference between Mouse CCNE2 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
(pg/mL)
O.D Average Corrected
20002.362
2.422
2.3922.306
10001.63
1.64
1.6351.549
5000.938
0.936
0.9370.851
2500.458
0.458
0.4580.372
1250.268
0.244
0.2560.17
62.50.19
0.186
0.1880.102
31.250.131
0.145
0.1380.052
00.078
0.094
0.086--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse CCNE2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse CCNE2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)91.92205.95926.8392.30224.24853.01
Standard deviation6.289.4732.816.3310.0938.98
C V (%)6.834.603.546.864.504.57

Recovery

The recovery of Mouse CCNE2 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)90-10697
EDTA plasma (n=5)87-10193
Cell culture media (n=5)93-10798

Linearity

Samples were spiked with high concentrations of Mouse CCNE2 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 (%)86-9895-10996-109
Average (%)92101103
1:4Range (%)91-10580-9189-101
Average (%)968595
1:8Range (%)88-10381-9286-100
Average (%)958791
1:16Range (%)92-10684-9784-98
Average (%)998990

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

Mouse CCNE2 (Cyclin-E2) ELISA Kit (MOES01652) 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.

Mouse CCNE2 (Cyclin-E2) ELISA Kit (MOES01652) Protein Information

UniProt Protein Function:CCNE2: Essential for the control of the cell cycle at the late G1 and early S phase. Interacts with the CDK2 (in vivo) and CDK3 (in vitro) protein kinases to form a serine/threonine kinase holoenzyme complex. The cyclin subunit imparts substrate specificity to the complex. Activated by papilloma viral oncoproteins E6 and E7 which bind to and inactivate p53 and Rb, respectively. According to PubMed:9858585, highest levels of expression in adult testis, thymus and brain. Lower levels in placenta, spleen and colon. Consistently elevated levels in tumor- derived cells compared to non-transformed proliferating cells. According to PubMed:9840927: low levels in thymus, prostate, brain, skeletal muscle, and kidney. Elevated levels in lung. According to PubMed:9840943 highly expressed in testis, placenta, thymus and brain. In a lesser extent in small intestine and colon. Belongs to the cyclin family. Cyclin E subfamily. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cell cycle regulation; Activator

Chromosomal Location of Human Ortholog: 8q22.1

Cellular Component: nucleoplasm; cytosol

Molecular Function:protein binding; cyclin-dependent protein kinase regulator activity

Biological Process: DNA replication initiation; cell division; mitotic cell cycle; regulation of cyclin-dependent protein kinase activity; cell cycle checkpoint; G1/S transition of mitotic cell cycle

NCBI Summary:The protein encoded by this gene belongs to 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. Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. This cyclin forms a complex with and functions as a regulatory subunit of CDK2. This cyclin has been shown to specifically interact with CIP/KIP family of CDK inhibitors, and plays a role in cell cycle G1/S transition. The expression of this gene peaks at the G1-S phase and exhibits a pattern of tissue specificity distinct from that of cyclin E1. A significantly increased expression level of this gene was observed in tumor-derived cells. [provided by RefSeq, Jul 2008]
UniProt Code:Q9Z238
NCBI GenInfo Identifier:17318565
NCBI Gene ID:9134
NCBI Accession:NP_477097.1
UniProt Secondary Accession:Q9Z238,Q7TMS8, Q9Z238,
UniProt Related Accession:O96020
Molecular Weight:47kDa
NCBI Full Name:G1/S-specific cyclin-E2
NCBI Synonym Full Names:cyclin E2
NCBI Official Symbol:CCNE2  
NCBI Official Synonym Symbols:CYCE2  
NCBI Protein Information:G1/S-specific cyclin-E2
UniProt Protein Name:G1/S-specific cyclin-E2
Protein Family:Regulatory protein
UniProt Gene Name:CCNE2  
UniProt Entry Name:CCNE2_HUMAN
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Additional Information

Reactivity:
Mouse
ELISA Type:
Sandwich
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