Human CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) CLIA Kit (HUES01204)

SKU:
HUES01204
€599

Description

ELISA Kit Technical ManualMSDS

Human CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) 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 CDKN2A . 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 CDKN2A 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 CDKN2A, 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 CDKN2A. The concentration of Human CDKN2A in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range125.00-8000 pg/mL
Sensitivity75.00 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human CDKN2A in samples. No significant cross-reactivity or interference between Human CDKN2A and analogues was observed.

Typical Data

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.

Concentration
(pg/mL)
RLU Average Corrected
800065209
77669
7143971418
400035326
35670
3549835477
200018719
16389
1755417533
10008380
8796
85888567
5004202
4012
41074086
2501982
1752
18671846
125.00724
770
747726
021
21
21--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CDKN2A were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CDKN2A were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)422.361244.973145.80381.151176.112885.29
Standard deviation41.31140.43360.8236.74130.43310.17
C V (%)9.7811.2811.479.6411.0910.75

Recovery

The recovery of Human CDKN2A 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)92-10599
EDTA plasma (n=5)98-110103
Cell culture media (n=5)90-10397

Linearity

Samples were spiked with high concentrations of Human CDKN2A 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 (%)86-9993-10697-111
Average (%)9399103
1:4Range (%)86-100101-11997-108
Average (%)91108102
1:8Range (%)99-11796-10991-102
Average (%)10710396
1:16Range (%)92-107100-11592-107
Average (%)9910699

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 CLIA 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 Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) CLIA Kit (HUES01204) 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 CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) CLIA Kit (HUES01204) Protein Information

UniProt Protein Function:Function: Acts as a negative regulator of the proliferation of normal cells by interacting strongly with CDK4 and CDK6. This inhibits their ability to interact with cyclins D and to phosphorylate the retinoblastoma protein. Ref.11 Ref.13
UniProt Protein Details:

Subunit structure: Heterodimer with CDK4 or CDK6. Predominant p16 complexes contained CDK6. Interacts (isoforms 1,2 and 4) with CDK4 (both 'T-172'-phosphorylated and non-phosphorylated forms); the interaction inhibits cyclin D-CDK4 kinase activity. Interacts with ISCO2. Ref.13 Ref.14

Subcellular location: Cytoplasm. Nucleus Ref.14.

Tissue specificity: Widely expressed but not detected in brain or skeletal muscle. Isoform 3 is pancreas-specific. Ref.2

Post-translational modification: Phosphorylation seems to increase interaction with CDK4.

Involvement in Disease: The association between cutaneous and uveal melanomas in some families suggests that mutations in CDKN2A may account for a proportion of uveal melanomas. However, CDKN2A mutations are rarely found in uveal melanoma patients.Melanoma, cutaneous malignant 2 (CMM2) [MIM:155601]: A malignant neoplasm of melanocytes, arising de novo or from a pre-existing benign nevus, which occurs most often in the skin but also may involve other sites.Note: Disease susceptibility is associated with variations affecting the gene represented in this entry. Ref.24 Ref.27 Ref.29 Ref.30 Ref.31 Ref.32 Ref.34 Ref.36 Ref.38 Ref.39 Ref.41 Ref.42Familial atypical multiple mole melanoma-pancreatic carcinoma syndrome (FAMMMPC) [MIM:606719]: An inherited cancer predisposition syndrome characterized by an increased risk of developing malignant melanoma and/or pancreatic cancer. Mutation carriers within families may develop either or both types of cancer.Note: The disease is caused by mutations affecting the gene represented in this entry.Li-Fraumeni syndrome (LFS) [MIM:151623]: Autosomal dominant familial cancer syndrome that in its classic form is defined by the existence of a proband affected by a sarcoma before 45 years with a first degree relative affected by any tumor before 45 years and another first degree relative with any tumor before 45 years or a sarcoma at any age. Other clinical definitions for LFS have been proposed (PubMed:8118819 and PubMed:8718514) and called Li-Fraumeni like syndrome (LFL). In these families affected relatives develop a diverse set of malignancies at unusually early ages. Four types of cancers account for 80% of tumors occurring in TP53 germline mutation carriers: breast cancers, soft tissue and bone sarcomas, brain tumors (astrocytomas) and adrenocortical carcinomas. Less frequent tumors include choroid plexus carcinoma or papilloma before the age of 15, rhabdomyosarcoma before the age of 5, leukemia, Wilms tumor, malignant phyllodes tumor, colorectal and gastric cancers.Note: The disease is caused by mutations affecting the gene represented in this entry. Ref.35Melanoma-astrocytoma syndrome (MASTS) [MIM:155755]: Characterized by a dual predisposition to melanoma and neural system tumors, commonly astrocytoma.Note: The disease is caused by mutations affecting the gene represented in this entry. Ref.37

Sequence similarities: Belongs to the CDKN2 cyclin-dependent kinase inhibitor family.Contains 4 ANK repeats.

Sequence caution: The sequence AAB60645.1 differs from that shown. Reason: Erroneous initiation.

NCBI Summary:This gene generates several transcript variants which differ in their first exons. At least three alternatively spliced variants encoding distinct proteins have been reported, two of which encode structurally related isoforms known to function as inhibitors of CDK4 kinase. The remaining transcript includes an alternate first exon located 20 Kb upstream of the remainder of the gene; this transcript contains an alternate open reading frame (ARF) that specifies a protein which is structurally unrelated to the products of the other variants. This ARF product functions as a stabilizer of the tumor suppressor protein p53 as it can interact with, and sequester, the E3 ubiquitin-protein ligase MDM2, a protein responsible for the degradation of p53. In spite of the structural and functional differences, the CDK inhibitor isoforms and the ARF product encoded by this gene, through the regulatory roles of CDK4 and p53 in cell cycle G1 progression, share a common functionality in cell cycle G1 control. This gene is frequently mutated or deleted in a wide variety of tumors, and is known to be an important tumor suppressor gene. [provided by RefSeq, Sep 2012]
UniProt Code:P42771
NCBI GenInfo Identifier:3041660
NCBI Gene ID:1029
NCBI Accession:P42771.2
UniProt Secondary Accession:P42771,O95440, Q15191, Q5VVJ5, Q96B52, Q9NP05, A5X2G7 D3DRK1,
UniProt Related Accession:P42771,Q8N726
Molecular Weight:
NCBI Full Name:Cyclin-dependent kinase inhibitor 2A, isoforms 1/2/3
NCBI Synonym Full Names:cyclin-dependent kinase inhibitor 2A
NCBI Official Symbol:CDKN2A
NCBI Official Synonym Symbols:ARF; MLM; P14; P16; P19; CMM2; INK4; MTS1; TP16; CDK4I; CDKN2; INK4A; MTS-1; P14ARF; P19ARF; P16INK4; P16INK4A; P16-INK4A
NCBI Protein Information:cyclin-dependent kinase inhibitor 2A; CDK4 inhibitor p16-INK4; multiple tumor suppressor 1; cell cycle negative regulator beta; cyclin-dependent kinase 4 inhibitor A; cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)
UniProt Protein Name:Cyclin-dependent kinase inhibitor 2A, isoforms 1/2/3
UniProt Synonym Protein Names:Cyclin-dependent kinase 4 inhibitor A; CDK4I; Multiple tumor suppressor 1; MTS-1; p16-INK4a
UniProt Gene Name:CDKN2A
UniProt Entry Name:CD2A1_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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