Mouse CCND2 (Cyclin-D2) CLIA Kit (MOES00213)

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SKU:
MOES00213
€649

Description

ELISA Kit Technical ManualMSDS

Mouse CCND2 (Cyclin-D2) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Mouse CCND2 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse CCND2 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 CCND2, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Mouse CCND2. The concentration of Mouse CCND2 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection methodChemiluminescence
Detection range12.50-800 pg/mL
Sensitivity7.50 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Mouse CCND2 in samples. No significant cross-reactivity or interference between Mouse CCND2 and analogues was observed.

Typical Data

As the RLU 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)
RLU Average Corrected
80051002
54282
5264252616
40019619
23849
2173421708
20010148
9268
97089682
1004332
4772
45524526
502343
2033
21882162
251083
1037
10601034
12.50489
529
509483
026
26
26--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse CCND2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse CCND2 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)38.2766.68355.8039.4970.76359.53
Standard deviation4.765.9439.145.018.1928.01
C V (%)12.448.9111.0012.6911.577.79

Recovery

The recovery of Mouse CCND2 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)86-10092
EDTA plasma (n=5)94-109102
Cell culture media (n=5)92-10398

Linearity

Samples were spiked with high concentrations of Mouse CCND2 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 (%)101-11693-10885-98
Average (%)10810091
1:4Range (%)91-10595-11392-109
Average (%)9610399
1:8Range (%)102-11588-10489-103
Average (%)1099694
1:16Range (%)100-11487-10099-112
Average (%)10694105

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 CLIA 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 Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Mouse CCND2 (Cyclin-D2) CLIA Kit (MOES00213) 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Mouse CCND2 (Cyclin-D2) CLIA Kit (MOES00213) Protein Information

UniProt Protein Function:CCND2: Regulatory component of the cyclin D2-CDK4 (DC) complex that phosphorylates and inhibits members of the retinoblastoma (RB) protein family including RB1 and regulates the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complex and the subsequent transcription of E2F target genes which are responsible for the progression through the G(1) phase. Hypophosphorylates RB1 in early G(1) phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also substrate for SMAD3, phosphorylating SMAD3 in a cell-cycle-dependent manner and repressing its transcriptional activity. Component of the ternary complex, cyclin D2/CDK4/p27Kip1, required for nuclear translocation and activity of the cyclin D-CDK4 complex. Interacts with either CDK4 or CDK6 protein kinase to form a serine/threonine kinase holoenzyme complex. The cyclin subunit imparts substrate specificity to the complex. Component of the ternary complex cyclin D/CDK4/p27Kip1 required for nuclear translocation and modulation of CDK4-mediated kinase activity. Belongs to the cyclin family. Cyclin D subfamily.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Cell cycle regulation

Cellular Component: chromatin; cyclin-dependent protein kinase holoenzyme complex; cytosol; nuclear membrane; nucleolus; nucleoplasm; nucleus

Molecular Function:protein binding; protein kinase binding

Biological Process: G1/S transition of mitotic cell cycle; positive regulation of cell proliferation; positive regulation of cyclin-dependent protein kinase activity; positive regulation of epithelial cell proliferation; positive regulation of protein amino acid phosphorylation; regulation of cell cycle

UniProt Code:P30280
NCBI GenInfo Identifier:231742
NCBI Gene ID:12444
NCBI Accession:P30280.1
UniProt Secondary Accession:P30280,Q04827,
UniProt Related Accession:P30280
Molecular Weight:32,897 Da
NCBI Full Name:G1/S-specific cyclin-D2
NCBI Synonym Full Names:cyclin D2
NCBI Official Symbol:Ccnd2  
NCBI Official Synonym Symbols:cD2; Vin1; Vin-1; C86853; AI256817; BF642806; 2600016F06Rik  
NCBI Protein Information:G1/S-specific cyclin-D2
UniProt Protein Name:G1/S-specific cyclin-D2
Protein Family:G1/S-specific cyclin
UniProt Gene Name:Ccnd2  
UniProt Entry Name:CCND2_MOUSE
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Additional Information

Reactivity:
Mouse
ELISA Type:
CLIA
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