Human Junction plakoglobin/gamma Catenin ELISA Kit (HUFI01473)

Product Name:	Human Junction plakoglobin/gamma Catenin ELISA Kit
Product Code:	HUFI01473
Size:	96 Assays
Alias:	JUP, Junction plakoglobin, Desmoplakin-3, Catenin gamma, Desmoplakin III
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human JUP concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human JUP and the recovery rates were calculated by comparing the measured value to the expected amount of Human JUP in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-98	93
EDTA plasma(n=5)	88-103	92
UFH plasma(n=5)	86-98	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human JUP and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-98%	88-103%	85-102%
EDTA plasma(n=5)	90-99%	82-99%	84-100%
UFH plasma(n=5)	80-96%	88-100%	82-90%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P14923

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

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 Âµl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 Âµl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Human Junction plakoglobin/gamma Catenin ELISA Kit (HUFI01473)

Description