Catenin-gamma Colorimetric Cell-Based ELISA Kit (CBCAB00566)

Product Name:	Catenin-gamma Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00566
ELISA Type:	Cell-Based
Target:	Catenin-gamma
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of Catenin-gamma concentration is achieved by an indirect ELISA format. In essence, Catenin-gamma is captured by Catenin-gamma-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: 3728, UniProt ID: P14923, OMIM: 173325/601214/611528, Unigene: Hs.514174
Gene Symbol:	JUP
Sub Type:	None

UniProt Protein Function:	CTNNG: Common junctional plaque protein. The membrane- associated plaques are architectural elements in an important strategic position to influence the arrangement and function of both the cytoskeleton and the cells within the tissue. The presence of plakoglobin in both the desmosomes and in the intermediate junctions suggests that it plays a central role in the structure and function of submembranous plaques. Acts as a substrate for VE-PTP and is required by it to stimulate VE- cadherin function in endothelial cells. Can replace beta-catenin in E-cadherin/catenin adhesion complexes which are proposed to couple cadherins to the actin cytoskeleton. Homodimer. Component of an E-cadherin/ catenin adhesion complex composed of at least E-cadherin/CDH1 and gamma- catenin/JUP, and possibly alpha-catenin/CTNNA1; the complex is located to adherens junctions. The stable association of CTNNA1 is controversial as CTNNA1 was shown not to bind to F-actin when assembled in the complex. Interacts with MUC1. Interacts with CAV1. Interacts with PTPRJ. Interacts with DSC2. Belongs to the beta-catenin family.
UniProt Protein Details:	Protein type:Cytoskeletal; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 17q21 Cellular Component: desmosome; internal side of plasma membrane; focal adhesion; apicolateral plasma membrane; intermediate filament; zonula adherens; catenin complex; intercellular junction; fascia adherens; cytosol; Z disc; actin cytoskeleton; cell-cell adherens junction; cytoskeleton; hemidesmosome; cytoplasm; plasma membrane; nucleus; lateral plasma membrane Molecular Function:protein binding; signal transducer activity; protein homodimerization activity; structural constituent of cell wall; cadherin binding; transcription coactivator activity; structural molecule activity; protein phosphatase binding; protein kinase binding; nuclear hormone receptor binding; alpha-catenin binding Biological Process: skin development; intercellular junction assembly and maintenance; cell migration; cytoskeletal anchoring; protein heterooligomerization; detection of mechanical stimulus; regulation of cell proliferation; cell-cell adhesion; positive regulation of protein import into nucleus; regulation of cell fate specification; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; vascular endothelial growth factor receptor signaling pathway Disease: Arrhythmogenic Right Ventricular Dysplasia, Familial, 12; Naxos Disease
NCBI Summary:	This gene encodes a major cytoplasmic protein which is the only known constituent common to submembranous plaques of both desmosomes and intermediate junctions. This protein forms distinct complexes with cadherins and desmosomal cadherins and is a member of the catenin family since it contains a distinct repeating amino acid motif called the armadillo repeat. Mutation in this gene has been associated with Naxos disease. Alternative splicing occurs in this gene; however, not all transcripts have been fully described. [provided by RefSeq, Jul 2008]
UniProt Code:	P14923
NCBI GenInfo Identifier:	205371866
NCBI Gene ID:	3728
NCBI Accession:	P14923.3
UniProt Secondary Accession:	P14923,Q15093, Q15151, Q7L3S5, Q86W21, Q9BWC4, Q9HCX9
UniProt Related Accession:	P14923
Molecular Weight:	745
NCBI Full Name:	Junction plakoglobin
NCBI Synonym Full Names:	junction plakoglobin
NCBI Official Symbol:	JUP
NCBI Official Synonym Symbols:	DP3; PDGB; PKGB; CTNNG; DPIII; ARVD12
NCBI Protein Information:	junction plakoglobin; desmoplakin-3; desmoplakin III; catenin (cadherin-associated protein), gamma 80kDa
UniProt Protein Name:	Junction plakoglobin
UniProt Synonym Protein Names:	Catenin gamma; Desmoplakin III; Desmoplakin-3
Protein Family:	Junction plakoglobin
UniProt Gene Name:	JUP
UniProt Entry Name:	PLAK_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-Catenin-gamma 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.)