Human CDKN2A/p16INK4a ELISA Kit (HUFI00802)

Product Name:	Human CDKN2A/p16INK4a ELISA Kit
Product Code:	HUFI00802
Size:	96 Assays
Alias:	CDKN2A, Cyclin Dependent Kinase Inhibitor 2A, p16INK4a, ARF, CDK4I, INK4a, MLM, p14ARF, p16, p19ARF, ARF, CDK4 inhibitor p16-INK4, CDKN2, cell cycle negative regulator beta, CMM2P16-INK4A, Cyclin-dependent kinase 4 inhibitor A, cyclin-dependent kinase inhibitor 2A, INK4, INK4A, MLMP16INK4, MTS-1, MTS1P14
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CDKN2A concentrations in serum plasma and other biological fluids.
Sensitivity:	0.375ng/ml
Range:	0.625-40ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human CDKN2A and the recovery rates were calculated by comparing the measured value to the expected amount of Human CDKN2A in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-97	93
EDTA plasma(n=5)	89-102	94
UFH plasma(n=5)	89-102	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CDKN2A and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-97%	87-102%	85-103%
EDTA plasma(n=5)	83-100%	83-100%	82-95%
UFH plasma(n=5)	83-98%	83-94%	81-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P42771

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 inDisease: 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

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.