Rat CDK4 (Cyclin Dependent Kinase 4) ELISA Kit (RTES00921)
- SKU:
- RTES00921
- Product type:
- ELISA Kit
- Reactivity:
- Rat
- ELISA Type:
- Sandwich
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| Detection Method: | Colormetric |
| Detection Range: | 78.13-5000 pg/mL |
| Sensitivity: | 46.88 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Rat CDK4 in samples. No significant cross-reactivity or interference between Rat CDK4 and analogues was observed. |
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 Rat 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 Rat 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 Rat 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 Rat CDK4. The concentration of Rat CDK4 in samples can be calculated by comparing the OD of the samples to the standard curve.
| 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; Cell cycle regulation; EC 2. 7. 11. 22; Protein kinase, CMGC; 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 complex binding; protein kinase activity Biological Process: circadian rhythm; G1/S transition of mitotic cell cycle; lens development in camera-type eye; organ regeneration; 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 hyperoxia; response to lead ion; response to organic substance; response to testosterone stimulus; response to toxin; signal transduction |
| NCBI Summary: | positive regulator of the cell cycle; plays a role in cell cycle progression [RGD, Feb 2006] |
| UniProt Code: | P35426 |
| NCBI GenInfo Identifier: | 543972 |
| NCBI Gene ID: | 94201 |
| NCBI Accession: | P35426. 1 |
| UniProt Related Accession: | P35426 |
| Molecular Weight: | 33,799 Da |
| NCBI Full Name: | Cyclin-dependent kinase 4 |
| NCBI Synonym Full Names: | cyclin-dependent kinase 4 |
| NCBI Official Symbol: | Cdk4 |
| NCBI Protein Information: | cyclin-dependent 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_RAT |
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 |
| 5000 | 2.542 2.59 | 2.566 | 2.504 |
| 2500 | 1.78 1.8 | 1.79 | 1.728 |
| 1250 | 0.992 0.954 | 0.973 | 0.911 |
| 625 | 0.52 0.54 | 0.53 | 0.468 |
| 312.5 | 0.258 0.25 | 0.254 | 0.192 |
| 156.25 | 0.181 0.165 | 0.173 | 0.111 |
| 78.13 | 0.117 0.119 | 0.118 | 0.056 |
| 0 | 0.061 0.063 | 0.062 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat CDK4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat CDK4 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 262.59 | 439.15 | 2142.00 | 263.69 | 478.34 | 2339.26 |
| Standard deviation | 15.41 | 19.54 | 117.17 | 13.26 | 26.74 | 90.06 |
| C V (%) | 5.87 | 4.45 | 5.47 | 5.03 | 5.59 | 3.85 |
Recovery
The recovery of Rat 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) | 87-98 | 93 |
| EDTA plasma (n=5) | 90-101 | 96 |
| Cell culture media (n=5) | 93-111 | 101 |
Linearity
Samples were spiked with high concentrations of Rat 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) | ||
| 1:2 | Range (%) | 89-103 | 90-102 | 91-105 |
| Average (%) | 96 | 96 | 98 | |
| 1:4 | Range (%) | 92-103 | 83-94 | 83-94 |
| Average (%) | 97 | 89 | 88 | |
| 1:8 | Range (%) | 91-105 | 80-92 | 82-95 |
| Average (%) | 96 | 87 | 87 | |
| 1:16 | Range (%) | 91-106 | 82-94 | 84-96 |
| Average (%) | 97 | 89 | 89 |
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 ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -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 (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
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