Product Name:	Human CDKN1B(Cyclin Dependent Kinase Inhibitor 1B) ELISA Kit
Product Code:	HUFI03173
Size:	96 Assays
Alias:	CDKN1B, CDKN4, KIP1, MEN1B, MEN4, P27, P27; KIP1, P27KIP1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CDKN1B 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 CDKN1B and the recovery rates were calculated by comparing the measured value to the expected amount of Human CDKN1B in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-97	90
EDTA plasma(n=5)	85-105	91
UFH plasma(n=5)	87-100	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CDKN1B 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)	85-104%	88-103%	85-102%
EDTA plasma(n=5)	90-96%	82-97%	89-101%
UFH plasma(n=5)	85-100%	84-100%	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

P46527

UniProt Protein Function:	p27Kip1: a cell-cycle regulatory protein that Interacts with cyclin-CDK2 and -CDK4, inhibiting cell cycle progression at G1. May mediate TGF beta-induced g1 arrest. Its degradation is triggered by its CDK dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state.
UniProt Protein Details:	Protein type:Protein kinase, regulatory subunit; Cell cycle regulation; Oncoprotein; Inhibitor Chromosomal Location of Human Ortholog: 12p13.1-p12 Cellular Component: nucleoplasm; cytoplasm; nucleus; cytosol; endosome Molecular Function:cyclin-dependent protein kinase inhibitor activity; protein binding; chaperone binding; protein complex binding; caspase activator activity; Hsp70 protein binding; protein phosphatase binding; transforming growth factor beta receptor, cytoplasmic mediator activity Biological Process: negative regulation of kinase activity; response to peptide hormone stimulus; nerve growth factor receptor signaling pathway; positive regulation of microtubule polymerization; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; response to estradiol stimulus; negative regulation of cell proliferation; sensory perception of sound; positive regulation of cell proliferation; response to glucose stimulus; negative regulation of mitotic cell cycle; cell cycle arrest; inner ear development; potassium ion transport; epidermal growth factor receptor signaling pathway; caspase activation; response to drug; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; negative regulation of cyclin-dependent protein kinase activity; negative regulation of cell motility; response to amino acid stimulus; response to cadmium ion; response to hypoxia; positive regulation of protein catabolic process; autophagic cell death; innate immune response; negative regulation of phosphorylation; negative regulation of cell growth; regulation of cyclin-dependent protein kinase activity; mitotic cell cycle; negative regulation of transcription, DNA-dependent; negative regulation of apoptosis; G1/S transition of mitotic cell cycle Disease: Multiple Endocrine Neoplasia, Type Iv
NCBI Summary:	This gene encodes a cyclin-dependent kinase inhibitor, which shares a limited similarity with CDK inhibitor CDKN1A/p21. The encoded protein binds to and prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus controls the cell cycle progression at G1. The degradation of this protein, which is triggered by its CDK dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state. Mutations in this gene are associated with multiple endocrine neoplasia type IV (MEN4). [provided by RefSeq, Apr 2014]
UniProt Code:	P46527
NCBI GenInfo Identifier:	1168871
NCBI Gene ID:	1027
NCBI Accession:	P46527.1
UniProt Secondary Accession:	P46527,Q16307, Q5U0H2, Q9BUS6,
UniProt Related Accession:	P46527
Molecular Weight:	Calculated: 22kDaObserved: 26kDa
NCBI Full Name:	Cyclin-dependent kinase inhibitor 1B
NCBI Synonym Full Names:	cyclin-dependent kinase inhibitor 1B (p27, Kip1)
NCBI Official Symbol:	CDKN1B
NCBI Official Synonym Symbols:	KIP1; MEN4; CDKN4; MEN1B; P27KIP1
NCBI Protein Information:	cyclin-dependent kinase inhibitor 1B
UniProt Protein Name:	Cyclin-dependent kinase inhibitor 1B
UniProt Synonym Protein Names:	Cyclin-dependent kinase inhibitor p27; p27Kip1
Protein Family:	Cyclin-dependent kinase inhibitor
UniProt Gene Name:	CDKN1B
UniProt Entry Name:	CDN1B_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.