|Detection Range:||15.63-1000 pg/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Human P27 in samples. No significant cross-reactivity or interference between Human P27 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 Human P27. 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 Human P27 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 P27, 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 Human P27. The concentration of Human P27 in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||p27Kip1: a cell-cycle regulatory protein that Interacts with cyclin-CDK2 and -CDK4, inhibiting cell cycle progression at G1. May mediate TGF beta-induced g1 arrest. Its degradation is triggered by its CDK dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state.|
|UniProt Protein Details:|
Protein type:Protein kinase, regulatory subunit; Cell cycle regulation; Oncoprotein; Inhibitor
Chromosomal Location of Human Ortholog: 12p13. 1-p12
Cellular Component: nucleoplasm; cytoplasm; nucleus; cytosol; endosome
Molecular Function:cyclin-dependent protein kinase inhibitor activity; protein binding; chaperone binding; protein complex binding; caspase activator activity; Hsp70 protein binding; protein phosphatase binding; transforming growth factor beta receptor, cytoplasmic mediator activity
Biological Process: negative regulation of kinase activity; response to peptide hormone stimulus; nerve growth factor receptor signaling pathway; positive regulation of microtubule polymerization; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; response to estradiol stimulus; negative regulation of cell proliferation; sensory perception of sound; positive regulation of cell proliferation; response to glucose stimulus; negative regulation of mitotic cell cycle; cell cycle arrest; inner ear development; potassium ion transport; epidermal growth factor receptor signaling pathway; caspase activation; response to drug; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; negative regulation of cyclin-dependent protein kinase activity; negative regulation of cell motility; response to amino acid stimulus; response to cadmium ion; response to hypoxia; positive regulation of protein catabolic process; autophagic cell death; innate immune response; negative regulation of phosphorylation; negative regulation of cell growth; regulation of cyclin-dependent protein kinase activity; mitotic cell cycle; negative regulation of transcription, DNA-dependent; negative regulation of apoptosis; G1/S transition of mitotic cell cycle
Disease: Multiple Endocrine Neoplasia, Type Iv
|NCBI Summary:||This gene encodes a cyclin-dependent kinase inhibitor, which shares a limited similarity with CDK inhibitor CDKN1A/p21. The encoded protein binds to and prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus controls the cell cycle progression at G1. The degradation of this protein, which is triggered by its CDK dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state. Mutations in this gene are associated with multiple endocrine neoplasia type IV (MEN4). [provided by RefSeq, Apr 2014]|
|NCBI GenInfo Identifier:||1168871|
|NCBI Gene ID:||1027|
|NCBI Accession:||P46527. 1|
|UniProt Secondary Accession:||P46527,Q16307, Q5U0H2, Q9BUS6,|
|UniProt Related Accession:||P46527|
|Molecular Weight:||Calculated: 22kDaObserved: 26kDa|
|NCBI Full Name:||Cyclin-dependent kinase inhibitor 1B|
|NCBI Synonym Full Names:||cyclin-dependent kinase inhibitor 1B (p27, Kip1)|
|NCBI Official Symbol:||CDKN1B|
|NCBI Official Synonym Symbols:||KIP1; MEN4; CDKN4; MEN1B; P27KIP1|
|NCBI Protein Information:||cyclin-dependent kinase inhibitor 1B|
|UniProt Protein Name:||Cyclin-dependent kinase inhibitor 1B|
|UniProt Synonym Protein Names:||Cyclin-dependent kinase inhibitor p27; p27Kip1|
|Protein Family:||Cyclin-dependent kinase inhibitor|
|UniProt Gene Name:||CDKN1B|
|UniProt Entry Name:||CDN1B_HUMAN|
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human P27 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human P27 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||6.88||4.55||3.84||5.31||4.71||5.28|
The recovery of Human P27 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)||86-101||92|
|Cell culture media (n=5)||91-105||99|
Samples were spiked with high concentrations of Human P27 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)|
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 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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 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.
- 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.
- Add 100 µL 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 90 µL 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.