Product Name:	Human Connexin 43 / GJA1 ELISA Kit
Product Code:	HUFI02371
Size:	96 Assays
Alias:	CX43, GJA1, GJAL, ODOD, SDTY3, connexin 43, connexin-43, Connexin-43, Cx43, CX43gap junction protein, alpha-like, DFNB38, Gap junction 43 kDa heart protein, gap junction alpha-1 protein, gap junction protein, alpha 1, 43kDa, gap junction protein, alpha 1, 43kDa, connexin 43, GJAL, HSS, ODD, SDTY3
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CX43 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 CX43 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CX43 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	90-104	97
EDTA plasma(n=5)	85-105	97
UFH plasma(n=5)	90-99	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CX43 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)	85-104%	91-105%	86-102%
EDTA plasma(n=5)	82-101%	84-100%	86-96%
UFH plasma(n=5)	88-100%	82-99%	83-99%

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

P17302

UniProt Protein Function:	GJA1: an integral membrane protein of the connexin family, alpha-type (group II) subfamily. Hexamers of connexin-43 form connexons, which aggregate together to form gap junctions, through which materials of low MW diffuse from one cell to a neighboring cell. May play a critical role in the physiology of hearing by participating in the recycling of potassium to the cochlear endolymph.
UniProt Protein Details:	Protein type:Membrane protein, integral; Channel, misc.; Motility/polarity/chemotaxis; Membrane protein, multi-pass Chromosomal Location of Human Ortholog: 6q22.31 Cellular Component: Golgi apparatus; endoplasmic reticulum membrane; focal adhesion; contractile fiber; integral to plasma membrane; lysosome; early endosome; intermediate filament; fascia adherens; cytosol; lipid raft; Golgi membrane; multivesicular body; connexon complex; mitochondrial outer membrane; apical plasma membrane; gap junction; plasma membrane; lateral plasma membrane Molecular Function:protein binding; signal transducer activity; ion transmembrane transporter activity; beta-tubulin binding; SH3 domain binding; gap junction channel activity; receptor binding; PDZ domain binding Biological Process: lens development in camera-type eye; response to peptide hormone stimulus; apoptosis; heart development; neuron migration; milk ejection; positive regulation of vasodilation; signal transduction; elevation of cytosolic calcium ion concentration; muscle contraction; cell-cell signaling; negative regulation of cardiac muscle cell proliferation; positive regulation of glomerular filtration; transport; response to glucose stimulus; positive regulation of striated muscle development; heart looping; ATP transport; adult heart development; chronic inflammatory response; positive regulation of I-kappaB kinase/NF-kappaB cascade; gap junction assembly; in utero embryonic development; epithelial cell maturation; regulation of bone remodeling; positive regulation of insulin secretion; skeletal muscle regeneration; regulation of calcium ion transport; vascular transport; protein oligomerization; osteoblast differentiation; positive regulation of osteoblast differentiation; negative regulation of endothelial cell proliferation; positive regulation of vasoconstriction; positive regulation of protein catabolic process; blood vessel morphogenesis; embryonic digit morphogenesis; regulation of bone mineralization; neurite morphogenesis; response to pH Disease: Syndactyly, Type Iii; Craniometaphyseal Dysplasia, Autosomal Recessive; Palmoplantar Keratoderma And Congenital Alopecia 1; Atrioventricular Septal Defect 3; Oculodentodigital Dysplasia; Erythrokeratodermia Variabilis Et Progressiva; Oculodentodigital Dysplasia, Autosomal Recessive; Hypoplastic Left Heart Syndrome 1
NCBI Summary:	This gene is a member of the connexin gene family. The encoded protein is a component of gap junctions, which are composed of arrays of intercellular channels that provide a route for the diffusion of low molecular weight materials from cell to cell. The encoded protein is the major protein of gap junctions in the heart that are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. A related intronless pseudogene has been mapped to chromosome 5. Mutations in this gene have been associated with oculodentodigital dysplasia, autosomal recessive craniometaphyseal dysplasia and heart malformations. [provided by RefSeq, May 2014]
UniProt Code:	P17302
NCBI GenInfo Identifier:	117706
NCBI Gene ID:	2697
NCBI Accession:	P17302.2
UniProt Secondary Accession:	P17302,Q6FHU1, Q9Y5I8, B2R5U9,
UniProt Related Accession:	P17302
Molecular Weight:	382
NCBI Full Name:	Gap junction alpha-1 protein
NCBI Synonym Full Names:	gap junction protein, alpha 1, 43kDa
NCBI Official Symbol:	GJA1
NCBI Official Synonym Symbols:	HSS; CMDR; CX43; GJAL; ODDD; AVSD3; HLHS1
NCBI Protein Information:	gap junction alpha-1 protein; connexin 43; connexin-43; gap junction 43 kDa heart protein
UniProt Protein Name:	Gap junction alpha-1 protein
UniProt Synonym Protein Names:	Connexin-43; Cx43; Gap junction 43 kDa heart protein
Protein Family:	Gap junction alpha-1 protein
UniProt Gene Name:	GJA1
UniProt Entry Name:	CXA1_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 µl 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 µl 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.

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Human Connexin 43 / GJA1 ELISA Kit

Description