Human AXIN2 (Axis Inhibition Protein 2) CLIA Kit (HUES00375)

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SKU:
HUES00375
€599

Description

ELISA Kit Technical ManualMSDS

Human AXIN2 (Axis Inhibition Protein 2) 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 Human AXIN2 . 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 Human AXIN2 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 Human AXIN2, 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 Human AXIN2. The concentration of Human AXIN2 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range62.50-4000 pg/mL
Sensitivity37.50 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human AXIN2 in samples. No significant cross-reactivity or interference between Human AXIN2 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
400056105
62057
5908159046
200026200
29518
2785927824
100013934
13034
1348413449
5006134
7076
66056570
2503430
3056
32433208
1251644
1518
15811546
62.50728
782
755720
035
35
35--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human AXIN2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AXIN2 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)205.45505.011362.30217.66501.871251.42
Standard deviation23.5751.6684.1917.6752.6077.46
C V (%)11.4710.236.188.1210.486.19

Recovery

The recovery of Human AXIN2 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)89-10395
EDTA plasma (n=5)84-9690
Cell culture media (n=5)93-111101

Linearity

Samples were spiked with high concentrations of Human AXIN2 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 (%)84-9987-10293-106
Average (%)9093100
1:4Range (%)86-10288-10294-111
Average (%)9394102
1:8Range (%)94-10899-11196-109
Average (%)99105102
1:16Range (%)92-10396-11089-102
Average (%)9710495

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

Human AXIN2 (Axis Inhibition Protein 2) CLIA Kit (HUES00375) 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.

Human AXIN2 (Axis Inhibition Protein 2) CLIA Kit (HUES00375) Protein Information

UniProt Protein Function:axin 2: is a negative regulator of the Wnt pathway, which is critical in stem cell signaling, morphogenesis, the mesenchymal-epithelial transition, and many cancers. Axin functions as a tumor suppressor. Probably facilitates the phosphorylation of beta-catenin and APC by GSK3B, leading to their ubiquitination and subsequent proteolysis. Is downregulated during progression of esophageal squamous cell carcinoma. Axin1/2 mediate cross-talk between TGF-beta and Wnt signaling pathways.
UniProt Protein Details:

Protein type:Adaptor/scaffold

Chromosomal Location of Human Ortholog: 17q24.1

Cellular Component: nucleoplasm; microtubule cytoskeleton; Golgi apparatus; centrosome; cytoplasmic microtubule; cytoplasm; cytoplasmic membrane-bound vesicle; postsynaptic density; plasma membrane; cell cortex; cytosol; nucleus; beta-catenin destruction complex

Molecular Function:protein binding; enzyme binding; ubiquitin protein ligase binding; beta-catenin binding; protein kinase binding; GTPase activator activity

Biological Process: odontogenesis; negative regulation of cell proliferation; cell proliferation; intramembranous ossification; regulation of mismatch repair; mRNA stabilization; dorsal/ventral axis specification; negative regulation of osteoblast differentiation; positive regulation of protein amino acid phosphorylation; maintenance of DNA repeat elements; bone mineralization; positive regulation of GTPase activity

Disease: Oligodontia-colorectal Cancer Syndrome

NCBI Summary:The Axin-related protein, Axin2, presumably plays an important role in the regulation of the stability of beta-catenin in the Wnt signaling pathway, like its rodent homologs, mouse conductin/rat axil. In mouse, conductin organizes a multiprotein complex of APC (adenomatous polyposis of the colon), beta-catenin, glycogen synthase kinase 3-beta, and conductin, which leads to the degradation of beta-catenin. Apparently, the deregulation of beta-catenin is an important event in the genesis of a number of malignancies. The AXIN2 gene has been mapped to 17q23-q24, a region that shows frequent loss of heterozygosity in breast cancer, neuroblastoma, and other tumors. Mutations in this gene have been associated with colorectal cancer with defective mismatch repair. [provided by RefSeq, Jul 2008]
UniProt Code:Q9Y2T1
NCBI GenInfo Identifier:12643949
NCBI Gene ID:8313
NCBI Accession:Q9Y2T1.1
UniProt Secondary Accession:Q9Y2T1,Q3MJ88, Q9H3M6, Q9UH84,
UniProt Related Accession:Q9Y2T1
Molecular Weight:843
NCBI Full Name:Axin-2
NCBI Synonym Full Names:axin 2
NCBI Official Symbol:AXIN2  
NCBI Official Synonym Symbols:AXIL; ODCRCS  
NCBI Protein Information:axin-2; conductin; axin-like protein; axis inhibition protein 2
UniProt Protein Name:Axin-2
UniProt Synonym Protein Names:Axin-like protein; Axil; Axis inhibition protein 2; Conductin
Protein Family:Axin
UniProt Gene Name:AXIN2  
UniProt Entry Name:AXIN2_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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