Human CDK4 (Cyclin Dependent Kinase 4) 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 Human CDK4 . 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 Human 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 Human CDK4, 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 Human CDK4. The concentration of Human CDK4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|Detection range||62.50-4000 pg/mL|
|Sample type||Serum, plasma and other biological fluids|
|Repeatability||CV < 15%|
This kit recognizes Human CDK4 in samples. No significant cross-reactivity or interference between Human CDK4 and analogues was observed.
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CDK4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CDK4 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||8.14||11.00||7.69||11.95||11.08||9.94|
The recovery of Human CDK4 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||99-115||107|
|Cell culture media (n=5)||85-101||92|
Samples were spiked with high concentrations of Human 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)|
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.
|Micro CLIA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 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 Diluent||1 vial, 20 mL||4'C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25X)||1 vial, 30 mL|
|Substrate Reagent A||1 vial, 5 mL||4'C (shading light)|
|Substrate Reagent B||1 vial, 5 mL||4'C (shading light)|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Human CDK4 (Cyclin Dependent Kinase 4) CLIA Kit (HUES01201) 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 100 µL 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.
Human CDK4 (Cyclin Dependent Kinase 4) CLIA Kit (HUES01201) 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:EC 220.127.116.11; Kinase, protein; Protein kinase, CMGC; Cell cycle regulation; Protein kinase, Ser/Thr (non-receptor); CMGC group; CDK family; CDK4 subfamily; CDK/CDK4 subfamily
Chromosomal Location of Human Ortholog: 12q14
Cellular Component: nucleoplasm; transcription factor complex; nuclear membrane; tight junction; perinuclear region of cytoplasm; nucleolus; cyclin-dependent protein kinase holoenzyme complex; nucleus; chromatin; cytosol
Molecular Function:protein binding; cyclin binding; cyclin-dependent protein kinase activity; protein complex binding; cyclin-dependent protein kinase regulator activity; ATP binding
Biological Process: circadian rhythm; lens development in camera-type eye; response to drug; establishment and/or maintenance of chromatin architecture; organ regeneration; positive regulation of cell size; positive regulation of translation; positive regulation of apoptosis; response to toxin; response to testosterone stimulus; signal transduction; protein amino acid phosphorylation; positive regulation of fibroblast proliferation; response to hyperoxia; regulation of gene expression; cell division; response to lead ion; positive regulation of cell proliferation; mitotic cell cycle; regulation of protein kinase activity; G1/S transition of mitotic cell cycle
Disease: Melanoma, Cutaneous Malignant, Susceptibility To, 3
|NCBI Summary:||The protein encoded by this gene is a member of the Ser/Thr protein kinase family. This protein is highly similar to the gene products of S. cerevisiae cdc28 and S. pombe cdc2. It is a catalytic subunit of the protein kinase complex that is important for cell cycle G1 phase progression. The activity of this kinase is restricted to the G1-S phase, which is controlled by the regulatory subunits D-type cyclins and CDK inhibitor p16(INK4a). This kinase was shown to be responsible for the phosphorylation of retinoblastoma gene product (Rb). Mutations in this gene as well as in its related proteins including D-type cyclins, p16(INK4a) and Rb were all found to be associated with tumorigenesis of a variety of cancers. Multiple polyadenylation sites of this gene have been reported. [provided by RefSeq, Jul 2008]|
|NCBI GenInfo Identifier:||1168867|
|NCBI Gene ID:||1019|
|UniProt Secondary Accession:||P11802,O00576, Q6FG61, B2R9A0, B4DNF9,|
|UniProt Related Accession:||P11802|
|Molecular Weight:||Calculated MW: 20kDa/33kDaObserved MW: 34kDa|
|NCBI Full Name:||Cyclin-dependent kinase 4|
|NCBI Synonym Full Names:||cyclin-dependent kinase 4|
|NCBI Official Symbol:||CDK4|
|NCBI Official Synonym Symbols:||CMM3; PSK-J3|
|NCBI Protein Information:||cyclin-dependent kinase 4; cell division protein kinase 4|
|UniProt Protein Name:||Cyclin-dependent kinase 4|
|UniProt Synonym Protein Names:||Cell division protein kinase 4; PSK-J3|
|Protein Family:||Cyclin-dependent kinase|
|UniProt Gene Name:||CDK4|
|UniProt Entry Name:||CDK4_HUMAN|