Keratin 16 Colorimetric Cell-Based ELISA Kit (CBCAB00724)

Product Name:	Keratin 16 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00724
ELISA Type:	Cell-Based
Target:	Keratin 16
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The Keratin 16 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Keratin 16 protein expression profile in cells. The kit can be used for measuring the relative amounts of Keratin 16 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Keratin 16.

Qualitative determination of Keratin 16 concentration is achieved by an indirect ELISA format. In essence, Keratin 16 is captured by Keratin 16-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: 3868, UniProt ID: P08779, OMIM: 144200/148067/167200/613000, Unigene: Hs.655160
Gene Symbol:	KRT16
Sub Type:	None

UniProt Protein Function:	K16: a type I cytoskeletal keratin. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. There are two types of cytoskeletal and microfibrillar keratin: type I (acidic; 40-55 kDa) [K9 to K20] and type II (neutral to basic; 56-70 kDa) [K1 to K8]. Both a basic and an acidic keratin are required for filament assembly. Generally associates with K6. K16 and K17 are coexpressed only in pathological situations such as metaplasias and carcinomas of the uterine cervix and in psoriasis vulgaris.
UniProt Protein Details:	Protein type:Cytoskeletal Chromosomal Location of Human Ortholog: 17q21.2 Cellular Component: cytoskeleton; intermediate filament; nucleus Molecular Function:protein binding; structural constituent of cytoskeleton Biological Process: keratinocyte differentiation; cell proliferation; epidermis development; keratinization; morphogenesis of an epithelium; intermediate filament cytoskeleton organization and biogenesis; innate immune response; cytoskeleton organization and biogenesis; inflammatory response; negative regulation of cell migration; keratinocyte migration; hair cycle; aging Disease: Palmoplantar Keratoderma, Nonepidermolytic, Focal; Pachyonychia Congenita 1
NCBI Summary:	The protein encoded by this gene is a member of the keratin gene family. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. Most of the type I cytokeratins consist of acidic proteins which are arranged in pairs of heterotypic keratin chains and are clustered in a region of chromosome 17q12-q21. This keratin has been coexpressed with keratin 14 in a number of epithelial tissues, including esophagus, tongue, and hair follicles. Mutations in this gene are associated with type 1 pachyonychia congenita, non-epidermolytic palmoplantar keratoderma and unilateral palmoplantar verrucous nevus. [provided by RefSeq, Jul 2008]
UniProt Code:	P08779
NCBI GenInfo Identifier:	23503075
NCBI Gene ID:	3868
NCBI Accession:	P08779.4
UniProt Secondary Accession:	P08779,P30654, Q16402, Q9UBG8, A8K488,
UniProt Related Accession:	P08779
Molecular Weight:	473
NCBI Full Name:	Keratin, type I cytoskeletal 16
NCBI Synonym Full Names:	keratin 16
NCBI Official Symbol:	KRT16
NCBI Official Synonym Symbols:	K16; PC1; CK16; K1CP; NEPPK; FNEPPK; KRT16A
NCBI Protein Information:	keratin, type I cytoskeletal 16; CK-16; keratin-16; cytokeratin 16; cytokeratin-16; focal non-epidermolytic palmoplantar keratoderma
UniProt Protein Name:	Keratin, type I cytoskeletal 16
UniProt Synonym Protein Names:	Cytokeratin-16; CK-16; Keratin-16; K16
Protein Family:	Keratin
UniProt Gene Name:	KRT16
UniProt Entry Name:	K1C16_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 16 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.)