CKI-epsilon Colorimetric Cell-Based ELISA (CBCAB01019)

Product Name:	CKI-epsilon Colorimetric Cell-Based ELISA
Product Code:	CBCAB01019
ELISA Type:	Cell-Based
Target:	CKI-epsilon
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of CKI-epsilon concentration is achieved by an indirect ELISA format. In essence, CKI-epsilon is captured by CKI-epsilon-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 1454, UniProt ID: P49674, OMIM: 600863, Unigene: Hs.474833
Gene Symbol:	CSNK1E
Sub Type:	None

UniProt Protein Function:	CK1E: an ubiquitous protein kinase of the CK1 family. Together with Dvl-1 and Frat-1 activate the Wnt signaling pathway. Central component of the circadian clock. May act as a negative regulator of circadian rhythmicity by phosphorylating PER1 and PER2. May play a role in cell cycle progression. Two splice variant isoforms have been described. Component of the circadian core oscillator, which includes the CRY proteins, CLOCK, or NPAS2, BMAL1 or BMAL2, CK1-D and/or CK1-E, TIMELESS and the PER proteins. Interacts directly with PER1 and PER2 which may lead to their degradation. Interacts with SOCS3. Mutations in hamster and Drosophila orthologs have circadian rhythm phenotypes, and the circadian gene period (per) is a substrate in both human and fly. A coding SNP variant in human, which increases CK1 activity, is negatively associated with circadian disorder. LOF mutations and LOH seen in mammary ductal carcinoma.
UniProt Protein Details:	Protein type:Protein kinase, CK1; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; EC 2.7.11.1; CK1 group; CK1 family Chromosomal Location of Human Ortholog: 22q13.1 Cellular Component: cytoplasm; nucleus; cytosol; ribonucleoprotein complex Molecular Function:protein serine/threonine kinase activity; protein binding; ATP binding; protein kinase activity Biological Process: circadian rhythm; Wnt receptor signaling pathway; negative regulation of Wnt receptor signaling pathway; organelle organization and biogenesis; endocytosis; DNA repair; signal transduction; regulation of circadian rhythm; protein amino acid phosphorylation; regulation of cell shape; peptidyl-serine phosphorylation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; circadian regulation of gene expression; mitotic cell cycle; G2/M transition of mitotic cell cycle
NCBI Summary:	The protein encoded by this gene is a serine/threonine protein kinase and a member of the casein kinase I protein family, whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. The encoded protein is found in the cytoplasm as a monomer and can phosphorylate a variety of proteins, including itself. This protein has been shown to phosphorylate period, a circadian rhythm protein. Two transcript variants encoding the same protein have been found for this gene. [provided by RefSeq, Feb 2014]
UniProt Code:	P49674
NCBI GenInfo Identifier:	1346369
NCBI Gene ID:	1454
NCBI Accession:	P49674.1
UniProt Related Accession:	P49674
Molecular Weight:	416
NCBI Full Name:	Casein kinase I isoform epsilon
NCBI Synonym Full Names:	casein kinase 1, epsilon
NCBI Official Symbol:	CSNK1E
NCBI Official Synonym Symbols:	HCKIE; CKIepsilon
NCBI Protein Information:	casein kinase I isoform epsilon; CKIe; CKI-epsilon
UniProt Protein Name:	Casein kinase I isoform epsilon
Protein Family:	Casein kinase
UniProt Gene Name:	CSNK1E
UniProt Entry Name:	KC1E_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-CKI-epsilon 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.)