Product Name:	Mouse CDKN2A/p16INK4a ELISA Kit
Product Code:	MOFI00710
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
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse CDKN2A concentrations in serum plasma and other biological fluids.
Sensitivity:	23.438pg/ml
Range:	39.063-2500pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-104	97
EDTA plasma(n=5)	87-105	94
UFH plasma(n=5)	86-103	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse 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)	85-105%	90-103%	86-105%
EDTA plasma(n=5)	82-96%	88-101%	87-95%
UFH plasma(n=5)	81-98%	83-100%	86-99%

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

Q64364

UniProt Protein Function:	p16-INK4A iso3: a cell-cycle regulatory protein that interacts with CDK4 and CDK6, inhibiting their ability to interact with cyclins D. Inhibits the phosphorylation of the retinoblastoma protein by CDK4 or CDK6, and entry into the S phase of the cell cycle. The p16INK4A and p14ARF proteins are encoded by CDKN2A, a known tumour suppressor gene in multiple cancers. CDKN2A is inactivated in 72% of cases of lung squamous cell carcinoma: 21% by epigenetic silencing by methylation, 18% inactivating mutation, 4% by exon 1b skipping, and 29% by homozygous deletion. Defects in CDKN2A are the cause of familial atypical multiple mole melanoma-pancreatic carcinoma syndrome, Li-Fraumeni syndrome, and the melanoma-astrocytoma syndrome. The melanoma-astrocytoma syndrome is characterized by a dual predisposition to melanoma and neural system tumors, commonly astrocytoma. Four alternatively spliced p16 isoforms have been reported. Two alternatively spliced isoforms of ARF have been reported.
UniProt Protein Details:	Protein type:Cell cycle regulation; Nucleolus; Tumor suppressor Cellular Component: cytoplasm; granular component; mitochondrion; nuclear body; nucleolus; nucleoplasm; nucleus; protein complex Molecular Function:cyclin-dependent protein kinase inhibitor activity; NF-kappaB binding; p53 binding; protein binding; protein kinase binding; protein N-terminus binding; SUMO ligase activity; transcription factor activity; transcription factor binding; ubiquitin-protein ligase inhibitor activity Biological Process: aging; apoptotic mitochondrial changes; caspase activation; cell aging; cell cycle arrest; epidermis development; G1/S transition of mitotic cell cycle; glucose homeostasis; inhibition of NF-kappaB transcription factor; mitochondrial depolarization; mitochondrion degradation; negative regulation of B cell proliferation; negative regulation of cell cycle; negative regulation of cell growth; negative regulation of cell proliferation; negative regulation of cell-matrix adhesion; negative regulation of cyclin-dependent protein kinase activity; negative regulation of immature T cell proliferation in the thymus; negative regulation of mammary gland epithelial cell proliferation; negative regulation of phosphorylation; negative regulation of protein binding; negative regulation of protein kinase activity; negative regulation of transcription, DNA-dependent; negative regulation of ubiquitin-protein ligase activity; positive regulation of apoptosis; positive regulation of apoptosis involved in mammary gland involution; positive regulation of DNA damage response, signal transduction by p53 class mediator; positive regulation of protein sumoylation; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; protein destabilization; protein polyubiquitination; protein stabilization; regulation of cyclin-dependent protein kinase activity; regulation of G2/M transition of mitotic cell cycle; regulation of gene expression; regulation of nucleocytoplasmic transport; regulation of protein export from nucleus; regulation of protein stability; regulation of transcription, DNA-dependent; response to drug; response to organic cyclic substance; response to organic nitrogen; rRNA transcription; somatic stem cell division; somatic stem cell maintenance
UniProt Code:	Q64364
NCBI GenInfo Identifier:	77416386
NCBI Gene ID:	12578
NCBI Accession:	Q64364.1
UniProt Secondary Accession:	Q64364,Q4U255, Q9QXC7, Q9R051,
UniProt Related Accession:	Q64364,P51480
Molecular Weight:	17,941 Da
NCBI Full Name:	Tumor suppressor ARF
NCBI Synonym Full Names:	cyclin-dependent kinase inhibitor 2A
NCBI Official Symbol:	Cdkn2a
NCBI Official Synonym Symbols:	Arf; p16; MTS1; Pctr1; p19ARF; p16INK4a; p19; ARF-INK4a; INK4a-ARF; Ink4a/Arf; p16(INK4a)
NCBI Protein Information:	cyclin-dependent kinase inhibitor 2A
UniProt Protein Name:	Tumor suppressor ARF
UniProt Synonym Protein Names:	Alternative reading frame; ARF; Cyclin-dependent kinase inhibitor 2A; p19ARF
Protein Family:	CDKN2A-interacting protein
UniProt Gene Name:	Cdkn2a
UniProt Entry Name:	ARF_MOUSE

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

Step	Procedure
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.