Mouse CDK4 (Cyclin Dependent Kinase 4) ELISA Kit (MOES01640)

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MOES01640
€549

Description

ELISA Kit Technical ManualMSDS

Mouse CDK4 (Cyclin Dependent Kinase 4) 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 CDK4 . 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 CDK4 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 CDK4, 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 CDK4. The concentration of Mouse CDK4 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 Range62.50-4000 pg/mL
Sensitivity37.50 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Mouse CDK4 in samples. No significant cross-reactivity or interference between Mouse CDK4 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
40002.341
2.365
2.3532.263
20001.583
1.605
1.5941.504
10000.888
0.86
0.8740.784
5000.496
0.512
0.5040.414
2500.296
0.266
0.2810.191
1250.188
0.184
0.1860.096
62.500.137
0.143
0.140.05
00.087
0.093
0.09--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse CDK4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse CDK4 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)202.49375.681529.85193.39375.101437.73
Standard deviation10.2120.8154.6212.7422.3969.15
C V (%)5.045.543.576.595.974.81

Recovery

The recovery of Mouse CDK4 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)88-10093
EDTA plasma (n=5)92-10498
Cell culture media (n=5)93-10498

Linearity

Samples were spiked with high concentrations of Mouse CDK4 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 (%)95-10586-9994-108
Average (%)10091101
1:4Range (%)92-10788-10084-97
Average (%)989389
1:8Range (%)89-10380-9385-98
Average (%)948692
1:16Range (%)88-10087-9886-98
Average (%)949393

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 CDK4 (Cyclin Dependent Kinase 4) ELISA Kit (MOES01640) 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 CDK4 (Cyclin Dependent Kinase 4) ELISA Kit (MOES01640) Protein Information

UniProt Protein Function:CDK4: a protein kinase of the CDK family that is important for cell cycle G1 phase progression. Its activity is restricted to the G1-S phase. Controlled by the regulatory subunits D-type cyclins and CDK inhibitor p16(INK4a). Phosphorylates the retinoblastoma gene product (Rb). Point mutations found in somatic and familial melanoma. Amplified in sarcomas, glioma and lymphoma. Amplified, methylated or deleted in head and neck squamous cell carcinoma. Overexpression drives epithelial tumors in mice. Disruption makes mice resistant to cancer. Inhibitor: PD332991.
UniProt Protein Details:

Protein type:Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, CMGC; EC 2.7.11.22; Cell cycle regulation; CMGC group; CDK family; CDK4 subfamily; CDK/CDK4 subfamily

Cellular Component: chromatin; cyclin-dependent protein kinase holoenzyme complex; cytosol; nuclear membrane; nucleolus; nucleus; perinuclear region of cytoplasm; tight junction; transcription factor complex

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

Biological Process: G1/S transition of mitotic cell cycle; positive regulation of apoptosis; positive regulation of cell proliferation; positive regulation of cell size; positive regulation of fibroblast proliferation; positive regulation of translation; protein amino acid phosphorylation; regulation of cell cycle; regulation of cell proliferation; regulation of gene expression; response to drug; response to lead ion; response to organic substance; response to testosterone stimulus; signal transduction

UniProt Code:P30285
NCBI GenInfo Identifier:231727
NCBI Gene ID:12567
NCBI Accession:P30285.1
UniProt Related Accession:P30285
Molecular Weight:33,751 Da
NCBI Full Name:Cyclin-dependent kinase 4
NCBI Synonym Full Names:cyclin-dependent kinase 4
NCBI Official Symbol:Cdk4  
NCBI Official Synonym Symbols:Crk3  
NCBI Protein Information:cyclin-dependent kinase 4
UniProt Protein Name:Cyclin-dependent kinase 4
UniProt Synonym Protein Names:CRK3; Cell division protein kinase 4; PSK-J3
Protein Family:Cyclin-dependent kinase
UniProt Gene Name:Cdk4  
UniProt Entry Name:CDK4_MOUSE
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Additional Information

Reactivity:
Mouse
ELISA Type:
Sandwich
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