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Human Connexin 32 / GJB1 ELISA Kit

SKU:
HUFI02500
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P08034
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich
Synonyms:
GJBeta1
Reactivity:
Human
Research Area:
Cell Biology
€599
Frequently bought together:

Description

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Human Connexin 32/GJB1 ELISA Kit

The Human Connexin 32 (GJB1) ELISA Kit is specifically designed for the precise measurement of Connexin 32 levels in human samples, including serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, guaranteeing accurate and consistent results for various research purposes.Connexin 32, also known as GJB1, is a critical protein involved in the formation of gap junctions within cells, allowing for direct communication between neighboring cells. Dysregulation of Connexin 32 has been linked to various diseases, including Charcot-Marie-Tooth disease, a hereditary neuropathy affecting the peripheral nerves.

By accurately quantifying Connexin 32 levels, researchers can gain valuable insights into the role of this protein in disease pathogenesis and potentially identify new therapeutic targets. The Human Connexin 32 (GJB1) ELISA Kit is a valuable tool for advancing our understanding of gap junction biology and its implications for human health.

Product Name:Human Connexin 32 / GJB1 ELISA Kit
Product Code:HUFI02500
Size:96 Assays
Alias:GJbeta1
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human GJbeta1 concentrations in serum plasma and other biological fluids.
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human GJbeta1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human GJbeta1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)91-10497
EDTA plasma(n=5)87-10597
UFH plasma(n=5)85-10395
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human GJbeta1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-101%92-103%86-102%
EDTA plasma(n=5)82-93%84-98%87-100%
UFH plasma(n=5)87-97%87-96%86-96%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

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
UniprotP08034
UniProt Protein Function:GJB1: One gap junction consists of a cluster of closely packed pairs of transmembrane channels, the connexons, through which materials of low MW diffuse from one cell to a neighboring cell. Defects in GJB1 are the cause of Charcot-Marie-Tooth disease X-linked type 1 (CMTX1); also designated CMT- X. CMTX1 is a form of Charcot-Marie-Tooth disease, the most common inherited disorder of the peripheral nervous system. Charcot- Marie-Tooth disease is classified in two main groups on the basis of electrophysiologic properties and histopathology: primary peripheral demyelinating neuropathies characterized by severely reduced motor nerve conduction velocities (NCVs) (less than 38m/s) and segmental demyelination and remyelination, and primary peripheral axonal neuropathies characterized by normal or mildly reduced NCVs and chronic axonal degeneration and regeneration on nerve biopsy. CMTX1 has both demyelinating and axonal features. Central nervous system involvement may occur. Defects in GJB1 may contribute to the phenotype of Dejerine-Sottas syndrome (DSS); also known as Dejerine-Sottas neuropathy (DSN) or hereditary motor and sensory neuropathy III (HMSN3). DSS is a severe degenerating neuropathy of the demyelinating Charcot-Marie-Tooth disease category, with onset by age 2 years. DSS is characterized by motor and sensory neuropathy with very slow nerve conduction velocities, increased cerebrospinal fluid protein concentrations, hypertrophic nerve changes, delayed age of walking as well as areflexia. There are both autosomal dominant and autosomal recessive forms of Dejerine- Sottas syndrome. Belongs to the connexin family. Beta-type (group I) subfamily.
UniProt Protein Details:Protein type:Motility/polarity/chemotaxis; Membrane protein, multi-pass; Membrane protein, integral; Channel, misc.

Chromosomal Location of Human Ortholog: Xq13.1

Cellular Component: connexon complex; endoplasmic reticulum membrane; integral to membrane

Molecular Function:protein homodimerization activity

Biological Process: nervous system development; gap junction assembly; cell-cell signaling; transport

Disease: Charcot-marie-tooth Disease, X-linked Dominant, 1

NCBI Summary:This gene encodes a member of the gap junction protein family. The gap junction proteins are membrane-spanning proteins that assemble to form gap junction channels that facilitate the transfer of ions and small molecules between cells. According to sequence similarities at the nucleotide and amino acid levels, the gap junction proteins are divided into two categories, alpha and beta. Mutations in this gene cause X-linked Charcot-Marie-Tooth disease, an inherited peripheral neuropathy. Alternatively spliced transcript variants encoding the same protein have been found for this gene. [provided by RefSeq, Oct 2008]
UniProt Code:P08034
NCBI GenInfo Identifier:117688
NCBI Gene ID:2705
NCBI Accession:P08034.1
UniProt Secondary Accession:P08034,Q5U0S4, B2R8R2, D3DVV2,
UniProt Related Accession:P08034
Molecular Weight:32,025 Da
NCBI Full Name:Gap junction beta-1 protein
NCBI Synonym Full Names:gap junction protein, beta 1, 32kDa
NCBI Official Symbol:GJB1
NCBI Official Synonym Symbols:CMTX; CX32; CMTX1
NCBI Protein Information:gap junction beta-1 protein; connexin 32; connexin-32; GAP junction 28 kDa liver protein
UniProt Protein Name:Gap junction beta-1 protein
UniProt Synonym Protein Names:Connexin-32; Cx32; GAP junction 28 kDa liver protein
Protein Family:Gap junction beta-1 protein
UniProt Gene Name:GJB1
UniProt Entry Name:CXB1_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.

StepProtocol
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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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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