Mouse CX26 (Connexin 26) CLIA Kit (MOES00196)

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SKU:
MOES00196
€649

Description

ELISA Kit Technical ManualMSDS

Mouse CX26 (Connexin 26) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Mouse CX26 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse CX26 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse CX26, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Mouse CX26. The concentration of Mouse CX26 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection methodChemiluminescence
Detection range7.81-500 pg/mL
Sensitivity4.69 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Mouse CX26 in samples. No significant cross-reactivity or interference between Mouse CX26 and analogues was observed.

Typical Data

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(pg/mL)
RLU Average Corrected
50050924
52150
5153751508
25019974
22022
2099820969
1259706
9086
93969367
62.54295
4729
45124483
31.252443
2155
22992270
15.631337
1163
12501221
7.81721
759
740711
029
29
29--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse CX26 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse CX26 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)25.4962.42215.0826.9168.13195.69
Standard deviation3.316.1318.542.247.2914.58
C V (%)12.999.828.628.3210.707.45

Recovery

The recovery of Mouse CX26 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)97-111104
EDTA plasma (n=5)100-112105
Cell culture media (n=5)90-10497

Linearity

Samples were spiked with high concentrations of Mouse CX26 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

    Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)95-11390-10385-96
Average (%)1039791
1:4Range (%)84-9788-10388-102
Average (%)909495
1:8Range (%)94-11087-10094-106
Average (%)1029299
1:16Range (%)97-11294-109102-120
Average (%)105100109

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Mouse CX26 (Connexin 26) CLIA Kit (MOES00196) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Mouse CX26 (Connexin 26) CLIA Kit (MOES00196) Protein Information

UniProt Protein Function:GJB2: 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 GJB2 are the cause of deafness autosomal recessive type 1A (DFNB1A). DFNB1A is a form of sensorineural hearing loss. Sensorineural deafness results from damage to the neural receptors of the inner ear, the nerve pathways to the brain, or the area of the brain that receives sound information. Defects in GJB2 are the cause of deafness autosomal dominant type 3A (DFNA3A). Defects in GJB2 are a cause of Vohwinkel syndrome (VS). VS is an autosomal dominant disease characterized by hyperkeratosis, constriction on finger and toes and congenital deafness. Defects in GJB2 are a cause of palmoplantar keratoderma with deafness (PPKDFN). PPKDFN is an autosomal dominant disorder characterized by the association of palmoplantar hyperkeratosis with progressive, bilateral, high-frequency, sensorineural deafness. Defects in GJB2 are a cause of keratitis-ichthyosis- deafness syndrome (KID syndrome); an autosomal dominant form of ectodermal dysplasia. Ectodermal dysplasias (EDs) constitute a heterogeneous group of developmental disorders affecting tissues of ectodermal origin. EDs are characterized by abnormal development of two or more ectodermal structures such as hair, teeth, nails and sweat glands, with or without any additional clinical sign. Each combination of clinical features represents a different type of ectodermal dysplasia. KID syndrome is characterized by the association of hyperkeratotic skin lesions with vascularizing keratitis and profound sensorineural hearing loss. Clinical features include deafness, ichthyosis, photobia, absent or decreased eyebrows, sparse or absent scalp hair, decreased sweating and dysplastic finger and toenails. Defects in GJB2 are the cause of Bart-Pumphrey syndrome (BPS). BPS is an autosomal dominant disorder characterized by sensorineural hearing loss, palmoplantar keratoderma, knuckle pads, and leukonychia, It shows considerable phenotypic variability. Defects in GJB2 are the cause of ichthyosis hystrix-like with deafness syndrome (HID syndrome). HID syndrome is an autosomal-dominant inherited keratinizing disorder characterized by sensorineural deafness and spiky hyperkeratosis affecting the entire skin. HID syndrome is considered to differ from the similar KID syndrome in the extent and time of occurrence of skin symptoms and the severity of the associated keratitis. Belongs to the connexin family. Beta-type (group I) subfamily.
UniProt Protein Details:

Protein type:Membrane protein, multi-pass; Cell adhesion; Membrane protein, integral; Motility/polarity/chemotaxis

Cellular Component: connexon complex; membrane; cytoplasm; integral to membrane; plasma membrane; gap junction; cell junction; lateral plasma membrane

Molecular Function:protein binding; gap junction channel activity

Biological Process: sensory perception of sound; gap junction assembly; cell communication; transmembrane transport

UniProt Code:Q00977
NCBI GenInfo Identifier:6680011
NCBI Gene ID:14619
NCBI Accession:NP_032151.1
UniProt Related Accession:Q00977
Molecular Weight:26,411 Da
NCBI Full Name:gap junction beta-2 protein
NCBI Synonym Full Names:gap junction protein, beta 2
NCBI Official Symbol:Gjb2  
NCBI Official Synonym Symbols:Cx26; Cnx26; Gjb-2; AI325222  
NCBI Protein Information:gap junction beta-2 protein; connexin 26; connexin-26; gap junction membrane channel protein beta 2
UniProt Protein Name:Gap junction beta-2 protein
UniProt Synonym Protein Names:Connexin-26; Cx26
Protein Family:Gap junction beta-2 protein
UniProt Gene Name:Gjb2  
UniProt Entry Name:CXB2_MOUSE
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Additional Information

Reactivity:
Mouse
ELISA Type:
CLIA
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