p27 Kip1 (Phospho-Thr187)Colorimetric Cell-Based ELISA Kit (CBCAB01584)

Product Name:	p27 Kip1 (Phospho-Thr187)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01584
ELISA Type:	Cell-Based
Target:	p27 Kip1 (Phospho-Thr187)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The p27 Kip1 (Phospho-Thr187) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect p27 Kip1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated p27 Kip1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on p27 Kip1 phosphorylation.

Qualitative determination of p27 Kip1 (Phospho-Thr187) concentration is achieved by an indirect ELISA format. In essence, p27 Kip1 (Phospho-Thr187) is captured by p27 Kip1 (Phospho-Thr187)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 1027, UniProt ID: P46527, OMIM: 600778/610755, Unigene: Hs.238990
Gene Symbol:	CDKN1B
Sub Type:	Phospho

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

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

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

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.	Incubate the cells for overnight at 37 °C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-p27 Kip1 (Phospho-Thr187) Antibody, Anti-p27 Kip1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)