p57 Kip2 (Phospho-Thr310)Colorimetric Cell-Based ELISA Kit (CBCAB01432)

Product Name:	p57 Kip2 (Phospho-Thr310)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01432
ELISA Type:	Cell-Based
Target:	p57 Kip2 (Phospho-Thr310)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The p57 Kip2 (Phospho-Thr310) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect p57 Kip2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated p57 Kip2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on p57 Kip2 phosphorylation.

Qualitative determination of p57 Kip2 (Phospho-Thr310) concentration is achieved by an indirect ELISA format. In essence, p57 Kip2 (Phospho-Thr310) is captured by p57 Kip2 (Phospho-Thr310)-specific primary (1Ã¸) antibodies while the HRP-conjugated secondary (2Ã¸) antibodies bind the Fc region of the 1Ã¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ã¸ 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: 1028, UniProt ID: P49918, OMIM: 130650/600856, Unigene: Hs.106070
Gene Symbol:	CDKN1C
Sub Type:	Phospho

UniProt Protein Function:	p57Kip2: Potent tight-binding inhibitor of several G1 cyclin/CDK complexes (cyclin E-CDK2, cyclin D2-CDK4, and cyclin A-CDK2) and, to lesser extent, of the mitotic cyclin B-CDC2. Negative regulator of cell proliferation. May play a role in maintenance of the non- proliferative state throughout life. Expressed in the heart, brain, lung, skeletal muscle, kidney, pancreas and testis. High levels are seen in the placenta while low levels are seen in the liver. Belongs to the CDI family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Inhibitor; Tumor suppressor; Nuclear receptor co-regulator; Cell cycle regulation Chromosomal Location of Human Ortholog: 11p15.5 Cellular Component: cytoplasm; nucleus Molecular Function:cyclin-dependent protein kinase inhibitor activity; protein binding Biological Process: negative regulation of kinase activity; camera-type eye development; adrenal gland development; multicellular organism growth; positive regulation of transcription, DNA-dependent; regulation of mitotic cell cycle; positive regulation of transforming growth factor beta receptor signaling pathway; negative regulation of transcription from RNA polymerase II promoter; uterus development; neuron maturation; negative regulation of phosphorylation; myeloid cell differentiation; kidney development; cell cycle arrest; negative regulation of transcription, DNA-dependent; skeletal development; aging; negative regulation of epithelial cell proliferation Disease: Intrauterine Growth Retardation, Metaphyseal Dysplasia, Adrenal Hypoplasia Congenita, And Genital Anomalies; Beckwith-wiedemann Syndrome
NCBI Summary:	This gene is imprinted, with preferential expression of the maternal allele. The encoded protein is a tight-binding, strong inhibitor of several G1 cyclin/Cdk complexes and a negative regulator of cell proliferation. Mutations in this gene are implicated in sporadic cancers and Beckwith-Wiedemann syndorome, suggesting that this gene is a tumor suppressor candidate. Three transcript variants encoding two different isoforms have been found for this gene. [provided by RefSeq, Oct 2010]
UniProt Code:	P49918
NCBI GenInfo Identifier:	1705731
NCBI Gene ID:	1028
NCBI Accession:	P49918.1
UniProt Related Accession:	P49918
Molecular Weight:	316
NCBI Full Name:	Cyclin-dependent kinase inhibitor 1C
NCBI Synonym Full Names:	cyclin-dependent kinase inhibitor 1C (p57, Kip2)
NCBI Official Symbol:	CDKN1C
NCBI Official Synonym Symbols:	BWS; WBS; p57; BWCR; KIP2; p57Kip2
NCBI Protein Information:	cyclin-dependent kinase inhibitor 1C; cyclin-dependent kinase inhibitor p57
UniProt Protein Name:	Cyclin-dependent kinase inhibitor 1C
UniProt Synonym Protein Names:	Cyclin-dependent kinase inhibitor p57; p57Kip2
Protein Family:	Cyclin-dependent kinase inhibitor
UniProt Gene Name:	CDKN1C
UniProt Entry Name:	CDN1C_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-p57 Kip2 (Phospho-Thr310) Antibody, Anti-p57 Kip2 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.)