Keratin 8 (Phospho-Ser73)Colorimetric Cell-Based ELISA Kit (CBCAB01219)

Product Name:	Keratin 8 (Phospho-Ser73)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01219
ELISA Type:	Cell-Based
Target:	Keratin 8 (Phospho-Ser73)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Keratin 8 (Phospho-Ser73) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Keratin 8 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Keratin 8 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Keratin 8 phosphorylation.

Qualitative determination of Keratin 8 (Phospho-Ser73) concentration is achieved by an indirect ELISA format. In essence, Keratin 8 (Phospho-Ser73) is captured by Keratin 8 (Phospho-Ser73)-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: 3856, UniProt ID: P05787, OMIM: 148060/215600, Unigene: Hs.533782/Hs.708445
Gene Symbol:	KRT8
Sub Type:	Phospho

UniProt Protein Function:	K8: a type II cytoskeletal keratin. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. Phsophorylated by p38 kinase, regulating cellular keratin filament reorganization. Phosphorylation on serine residues is enhanced during EGF stimulation and mitosis. Mutation of this protein is a risk factor for cryptogenic liver failure.
UniProt Protein Details:	Protein type:Cytoskeletal Chromosomal Location of Human Ortholog: 12q13 Cellular Component: dystrophin-associated glycoprotein complex; nucleoplasm; intermediate filament cytoskeleton; nuclear matrix; costamere; cytoplasm; keratin filament; intermediate filament; intercellular junction; Z disc; nucleus; sarcolemma Molecular Function:protein binding; protein complex binding; structural molecule activity Biological Process: tumor necrosis factor-mediated signaling pathway; viral reproduction; response to other organism; sarcomere organization; response to hydrostatic pressure Disease: Cirrhosis, Familial
NCBI Summary:	This gene is a member of the type II keratin family clustered on the long arm of chromosome 12. Type I and type II keratins heteropolymerize to form intermediate-sized filaments in the cytoplasm of epithelial cells. The product of this gene typically dimerizes with keratin 18 to form an intermediate filament in simple single-layered epithelial cells. This protein plays a role in maintaining cellular structural integrity and also functions in signal transduction and cellular differentiation. Mutations in this gene cause cryptogenic cirrhosis. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Jan 2012]
UniProt Code:	P05787
NCBI GenInfo Identifier:	90110027
NCBI Gene ID:	3856
NCBI Accession:	P05787.7
UniProt Secondary Accession:	P05787,Q14099, Q14716, Q14717, Q53GJ0, Q6DHW5, Q6GMY0 Q6P4C7, Q96J60, A8K4H3, B0AZN5, F8VXB4,
UniProt Related Accession:	P05787
Molecular Weight:	483
NCBI Full Name:	Keratin, type II cytoskeletal 8
NCBI Synonym Full Names:	keratin 8
NCBI Official Symbol:	KRT8
NCBI Official Synonym Symbols:	K8; KO; CK8; CK-8; CYK8; K2C8; CARD2
NCBI Protein Information:	keratin, type II cytoskeletal 8; cytokeratin 8; cytokeratin-8; type-II keratin Kb8
UniProt Protein Name:	Keratin, type II cytoskeletal 8
UniProt Synonym Protein Names:	Cytokeratin-8; CK-8; Keratin-8; K8; Type-II keratin Kb8
Protein Family:	Keratin
UniProt Gene Name:	KRT8
UniProt Entry Name:	K2C8_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-Keratin 8 (Phospho-Ser73) Antibody, Anti-Keratin 8 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.)