Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human CDK2 in samples. No significant cross-reactivity or interference between Human CDK2 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 CDK2. 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 CDK2 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 CDK2, 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 CDK2. The concentration of Human CDK2 in samples can be calculated by comparing the OD of the samples to the standard curve.

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

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.

Concentration (ng/mL)	O.D	Average	Corrected
20	2.24 2.274	2.257	2.204
10	1.555 1.567	1.561	1.508
5	0.861 0.847	0.854	0.801
2.5	0.468 0.482	0.475	0.422
1.25	0.253 0.239	0.246	0.193
0.63	0.15 0.146	0.148	0.095
0.31	0.095 0.109	0.102	0.049
0	0.046 0.06	0.053	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CDK2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CDK2 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.93	2.50	9.86	0.85	2.28	9.95
Standard deviation	0.05	0.12	0.39	0.04	0.10	0.52
C V (%)	5.38	4.80	3.96	4.71	4.39	5.23

Recovery

The recovery of Human CDK2 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)	86-99	92
EDTA plasma (n=5)	88-98	93
Cell culture media (n=5)	90-107	97

Linearity

Samples were spiked with high concentrations of Human CDK2 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)
1:2	Range (%)	88-101	84-96	90-102
1:2	Average (%)	94	91	95
1:4	Range (%)	88-102	84-94	85-98
1:4	Average (%)	93	89	92
1:8	Range (%)	87-100	86-101	85-100
1:8	Average (%)	94	92	92
1:16	Range (%)	86-100	82-94	85-100
1:16	Average (%)	93	88	92

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 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 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl 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 100µL 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 350µL 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 100µL 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 90µL 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.