Product Name:	Human Cdk2 ELISA Kit
Product Code:	HUFI01666
Size:	96 Assays
Alias:	CDK2, Cyclin-dependent kinase 2, Cell division protein kinase 2, p33 protein kinase, CDKN2, cdc2-related protein kinase, Cell division kinase 2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CDK2 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human CDK2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CDK2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-102	92
EDTA plasma(n=5)	88-104	96
UFH plasma(n=5)	86-101	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CDK2 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)	89-96%	85-103%	88-102%
EDTA plasma(n=5)	82-97%	83-94%	82-101%
UFH plasma(n=5)	80-97%	88-98%	86-94%

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

P24941

UniProt Protein Function:	CDK2: a protein kinase of the CDK family. An important component of the cell cycle machinery. Activity of CDK2 is maximal during S phase and G2. Cdk2/cyclin E kinase activity is important for the G1 to S transition and phosphorylates the Rb protein. In S-phase, active cdk2/cyclin A complexes predominate and phosphorylate E2F, and the active cdk2 complex persists in the nucleus through G2. Part of the Rb pathway disregulated in most tumors. Target of several candidate cancer drugs. However, inhibition does not always prevent cancer cell growth, possibly due to CDK redundancy. Inhibitors: BMS-265246, BMS-265246-01, R-roscovitine (CYC200, CYC202), SU9516, R547, L868276
UniProt Protein Details:	Protein type:Kinase, protein; EC 2.7.11.22; Protein kinase, CMGC; Protein kinase, Ser/Thr (non-receptor); Cell cycle regulation; CMGC group; CDK family; CDK1 subfamily; CDK/CDK1 subfamily Chromosomal Location of Human Ortholog: 12q13 Cellular Component: Cajal body; centrosome; chromosome, telomeric region; X chromosome; condensed chromosome; cytosol; Y chromosome; nucleoplasm; transcription factor complex; cytoplasm; cyclin-dependent protein kinase holoenzyme complex; nucleus; endosome Molecular Function:cyclin binding; protein binding; cyclin-dependent protein kinase activity; metal ion binding; histone kinase activity; ATP binding Biological Process: G1 DNA damage checkpoint; meiosis; mitosis; positive regulation of transcription, DNA-dependent; histone phosphorylation; DNA repair; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; peptidyl-serine phosphorylation; regulation of ubiquitin-protein ligase activity during mitotic cell cycle; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; cell division; positive regulation of cell proliferation; Ras protein signal transduction; mitotic cell cycle; DNA replication; G2/M transition of mitotic cell cycle; blood coagulation; centrosome duplication; potassium ion transport; G1/S transition of mitotic cell cycle; positive regulation of DNA replication initiation
NCBI Summary:	This gene encodes a member of a family of serine/threonine protein kinases that participate in cell cycle regulation. The encoded protein is the catalytic subunit of the cyclin-dependent protein kinase complex, which regulates progression through the cell cycle. Activity of this protein is especially critical during the G1 to S phase transition. This protein associates with and regulated by other subunits of the complex including cyclin A or E, CDK inhibitor p21Cip1 (CDKN1A), and p27Kip1 (CDKN1B). Alternative splicing results in multiple transcript variants. [provided by RefSeq, Mar 2014]
UniProt Code:	P24941
NCBI GenInfo Identifier:	116051
NCBI Gene ID:	1017
NCBI Accession:	P24941.2
UniProt Secondary Accession:	P24941,O75100, A8K7C6,
UniProt Related Accession:	P24941
Molecular Weight:	30,035 Da
NCBI Full Name:	Cyclin-dependent kinase 2
NCBI Synonym Full Names:	cyclin-dependent kinase 2
NCBI Official Symbol:	CDK2
NCBI Official Synonym Symbols:	CDKN2; p33(CDK2)
NCBI Protein Information:	cyclin-dependent kinase 2; p33 protein kinase; cdc2-related protein kinase; cell division protein kinase 2
UniProt Protein Name:	Cyclin-dependent kinase 2
UniProt Synonym Protein Names:	Cell division protein kinase 2; p33 protein kinase
Protein Family:	Cyclin-dependent kinase
UniProt Gene Name:	CDK2
UniProt Entry Name:	CDK2_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.