Mouse CCND3 (Cyclin-D3) ELISA Kit (MOES00938)

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MOES00938
€549

Description

ELISA Kit Technical ManualMSDS

Mouse CCND3 (Cyclin-D3) 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 CCND3 . 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 CCND3 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 CCND3, 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 CCND3. The concentration of Mouse CCND3 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 CCND3 in samples. No significant cross-reactivity or interference between Mouse CCND3 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
20002.538
2.55
2.5442.477
10001.65
1.656
1.6531.586
5000.922
0.89
0.9060.839
2500.483
0.495
0.4890.422
1250.281
0.277
0.2790.212
62.50.181
0.157
0.1690.102
31.250.116
0.122
0.1190.052
00.063
0.071
0.067--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse CCND3 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse CCND3 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)95.63237.01745.9392.18239.43807.36
Standard deviation4.8213.9639.686.4211.4028.66
C V (%)5.045.895.326.964.763.55

Recovery

The recovery of Mouse CCND3 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)92-10698
EDTA plasma (n=5)88-10295
Cell culture media (n=5)84-9791

Linearity

Samples were spiked with high concentrations of Mouse CCND3 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 (%)97-11388-10490-102
Average (%)1059595
1:4Range (%)86-10085-9582-92
Average (%)929087
1:8Range (%)88-10283-9583-96
Average (%)948890
1:16Range (%)87-10384-9885-99
Average (%)949091

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 CCND3 (Cyclin-D3) ELISA Kit (MOES00938) 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 CCND3 (Cyclin-D3) ELISA Kit (MOES00938) Protein Information

UniProt Protein Function:CCND3: Regulatory component of the cyclin D3-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 D3/CDK4/p27Kip1, required for nuclear translocation and activity of the cyclin D-CDK4 complex. Interacts with the CDK4 and CDK6 protein kinases to form a serine/threonine kinase holoenzyme complex. The cyclin subunit imparts substrate specificity to the complex. Interacts with ATF5. Interacts with EIF3K. Component of the ternary complex cyclin D/CDK4/p27Kip1 required for nuclear translocation and modulation of CDK4-mediated kinase activity. Can form similar complexes with either p21Cip1 or CDKN2A. Belongs to the cyclin family. Cyclin D subfamily. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cell cycle regulation; Activator; Nuclear receptor co-regulator

Chromosomal Location of Human Ortholog: 6p21

Cellular Component: membrane; cytoplasm; cyclin-dependent protein kinase holoenzyme complex; nucleus

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

Biological Process: T cell proliferation; positive regulation of cyclin-dependent protein kinase activity; cell division; positive regulation of protein amino acid phosphorylation; cell cycle; signal transduction

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 CDK4 or CDK6, whose activtiy is required for cell cycle G1/S transition. This protein has been shown to interact with and be involved in the phosphorylation of tumor suppressor protein Rb. The CDK4 activity associated with this cyclin was reported to be necessary for cell cycle progression through G2 phase into mitosis after UV radiation. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2008]
UniProt Code:P30282
NCBI GenInfo Identifier:209862835
NCBI Gene ID:896
NCBI Accession:NP_001129489.1
UniProt Secondary Accession:P30282,P30282,
UniProt Related Accession:P30281
Molecular Weight:32kDa
NCBI Full Name:G1/S-specific cyclin-D3 isoform 1
NCBI Synonym Full Names:cyclin D3
NCBI Official Symbol:CCND3  
NCBI Protein Information:G1/S-specific cyclin-D3
UniProt Protein Name:G1/S-specific cyclin-D3
Protein Family:Protein
UniProt Gene Name:CCND3  
UniProt Entry Name:CCND3_HUMAN
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Additional Information

Reactivity:
Mouse
ELISA Type:
Sandwich
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