Product Name:	Human CDK1 ELISA Kit
Product Code:	HUFI02300
Size:	96 Assays
Alias:	CDK1, CDC28A, cell cycle controller CDC2, Cell division control protein 2 homolog, Cell division protein kinase 1, cyclin-dependent kinase 1, G1 to S and G2 to M, p34 protein kinase, P34CDC2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CDK1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human CDK1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CDK1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-105	94
EDTA plasma(n=5)	90-105	99
UFH plasma(n=5)	85-97	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CDK1 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)	89-105%	89-102%	88-98%
EDTA plasma(n=5)	82-96%	83-96%	91-97%
UFH plasma(n=5)	80-100%	80-100%	81-98%

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

P06493

UniProt Protein Function:	CDK1: a protein kinase of the CDK family. Catalytic subunit of the conserved protein complex known as M-phase promoting factor (MPF), which is essential for G1/S and G2/M phase transitions. Mitotic cyclins stably associate with this protein and function as regulatory subunits. Its activity is controlled by cyclin availability and phosphorylation through the cell cycle. Activated in many cancers including colon, liver and breast. The T isoform, which lacks a regulatory region, is expressed in breast cancer. Inhibition in cancer cells may drive cells into apoptosis. May also drive cell migration. Inhibitors: BMS-265246, BMS-265246-01. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell cycle regulation; EC 2.7.11.23; EC 2.7.11.22; Motility/polarity/chemotaxis; Kinase, protein; Protein kinase, CMGC; Protein kinase, Ser/Thr (non-receptor); CMGC group; CDK family; CDK/CDK1 subfamily; CDK1 subfamily Chromosomal Location of Human Ortholog: 10q21.1 Cellular Component: nucleoplasm; centrosome; membrane; mitochondrion; spindle microtubule; cytoplasm; midbody; nucleus; cytosol Molecular Function:RNA polymerase subunit kinase activity; protein serine/threonine kinase activity; protein binding; cyclin binding; cyclin-dependent protein kinase activity; histone kinase activity; Hsp70 protein binding; ATP binding; protein kinase activity Biological Process: regulation of Schwann cell differentiation; activation of MAPKK activity; nerve growth factor receptor signaling pathway; activation of MAPK activity; histone phosphorylation; response to toxin; ventricular cardiac muscle cell development; regulation of embryonic development; centrosome cycle; stress-activated MAPK cascade; toll-like receptor 3 signaling pathway; response to organic cyclic substance; toll-like receptor 5 signaling pathway; small GTPase mediated signal transduction; protein complex assembly; G2/M transition of mitotic cell cycle; toll-like receptor 4 signaling pathway; positive regulation of cardiac muscle cell proliferation; response to drug; mitosis; organ regeneration; fibroblast growth factor receptor signaling pathway; positive regulation of protein import into nucleus, translocation; cell aging; pronuclear fusion; toll-like receptor 2 signaling pathway; chromosome condensation; response to ethanol; cell division; response to activity; toll-like receptor 9 signaling pathway; response to amine stimulus; G1/S transition of mitotic cell cycle; negative regulation of apoptosis; positive regulation of ubiquitin-protein ligase activity during mitotic cell cycle; axon guidance; apoptosis; mitotic nuclear envelope disassembly; positive regulation of mitotic cell cycle; toll-like receptor 10 signaling pathway; epithelial cell differentiation; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; response to axon injury; DNA replication; epidermal growth factor receptor signaling pathway; cell migration; MyD88-independent toll-like receptor signaling pathway; organelle organization and biogenesis; MAPKKK cascade; peptidyl-threonine phosphorylation; microtubule cytoskeleton organization and biogenesis; DNA repair; MyD88-dependent toll-like receptor signaling pathway; G1/S-specific transcription in mitotic cell cycle; peptidyl-serine phosphorylation; response to cadmium ion; regulation of ubiquitin-protein ligase activity during mitotic cell cycle; response to copper ion; mitotic cell cycle G2/M transition DNA damage checkpoint; Ras protein signal transduction; insulin receptor signaling pathway; toll-like receptor signaling pathway; innate immune response; mitotic cell cycle; vascular endothelial growth factor receptor signaling pathway; positive regulation of DNA replication
NCBI Summary:	The protein encoded by this gene is a member of the Ser/Thr protein kinase family. This protein is a catalytic subunit of the highly conserved protein kinase complex known as M-phase promoting factor (MPF), which is essential for G1/S and G2/M phase transitions of eukaryotic cell cycle. Mitotic cyclins stably associate with this protein and function as regulatory subunits. The kinase activity of this protein is controlled by cyclin accumulation and destruction through the cell cycle. The phosphorylation and dephosphorylation of this protein also play important regulatory roles in cell cycle control. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Mar 2009]
UniProt Code:	P06493
NCBI GenInfo Identifier:	334302921
NCBI Gene ID:	983
NCBI Accession:	P06493.3
UniProt Secondary Accession:	P06493,O60764, A8K7C4, C9J497,
UniProt Related Accession:	P06493
Molecular Weight:	297
NCBI Full Name:	Cyclin-dependent kinase 1
NCBI Synonym Full Names:	cyclin-dependent kinase 1
NCBI Official Symbol:	CDK1
NCBI Official Synonym Symbols:	CDC2; CDC28A; P34CDC2
NCBI Protein Information:	cyclin-dependent kinase 1; p34 protein kinase; cell cycle controller CDC2; cell division protein kinase 1; cell division control protein 2 homolog; cell division cycle 2, G1 to S and G2 to M
UniProt Protein Name:	Cyclin-dependent kinase 1
UniProt Synonym Protein Names:	Cell division control protein 2 homolog; Cell division protein kinase 1; p34 protein kinase
UniProt Gene Name:	CDK1
UniProt Entry Name:	CDK1_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 Âµl 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 Âµl 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.