Human JAK2 (Janus Kinase 2) ELISA Kit (HUES03125)

SKU:
HUES03125
€539

Description

ELISA Kit Technical ManualMSDS

Human JAK2 (Janus Kinase 2) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human JAK2 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human JAK2 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 JAK2, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human JAK2. The concentration of Human JAK2 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range125.00-8000 pg/mL
Sensitivity75.00 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human JAK2 in samples. No significant cross-reactivity or interference between Human JAK2 and analogues was observed.

Typical Data

As the OD 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)
O.D Average Corrected
80002.583
2.625
2.6042.539
40001.753
1.793
1.7731.708
20001.069
1.063
1.0661.001
10000.537
0.549
0.5430.478
5000.321
0.295
0.3080.243
2500.189
0.173
0.1810.116
125.000.117
0.129
0.1230.058
00.06
0.07
0.065--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human JAK2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human JAK2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)402.55750.573976.07362.53678.443953.45
Standard deviation25.2431.82213.9125.0931.14124.53
C V (%)6.274.245.386.924.593.15

Recovery

The recovery of Human JAK2 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)93-10999
EDTA plasma (n=5)90-10195
Cell culture media (n=5)86-9993

Linearity

Samples were spiked with high concentrations of Human JAK2 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 (%)94-11097-11197-111
Average (%)102103103
1:4Range (%)89-10187-9883-99
Average (%)959290
1:8Range (%)89-10479-9186-101
Average (%)958593
1:16Range (%)91-10482-9688-103
Average (%)978894

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 ELISA 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 Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human JAK2 (Janus Kinase 2) ELISA Kit (HUES03125) 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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human JAK2 (Janus Kinase 2) ELISA Kit (HUES03125) Protein Information

UniProt Protein Function:JAK2: a non-receptor tyrosine-kinase involved in a specific subset of cytokine receptor signaling pathways, including IL-3, -5 and GM-CSF. Interacts with IL23R, SKB1 and STAM2. It has been found to be constitutively associated with the prolactin receptor and is required for responses to gamma interferon. Mice that do not express an active protein for this gene exhibit embryonic lethality associated with the absence of definitive erythropoiesis. Fusion of Jak2 to TEL1 (ETV6) by t(9;12)(p24;p13) causes myeloproliferative disease in humans and mouse models. The Jak inhibitor AG490 inhibits constitutive Jak2 phosphorylation and causes apoptosis in cells from breast cancer and relapsing acute lymphoblastic leukemia. A single activating mutation is associated with several hematological malignancies. Inhibitor: AG490.
UniProt Protein Details:

Protein type:Protein kinase, TK; EC 2.7.10.2; Kinase, protein; Oncoprotein; Protein kinase, tyrosine (non-receptor); TK group; JakA family

Chromosomal Location of Human Ortholog: 9p24

Cellular Component: caveola; cytoplasm; cytoskeleton; cytosol; extrinsic to internal side of plasma membrane; lipid raft; nuclear matrix; nucleoplasm; nucleus

Molecular Function:acetylcholine receptor binding; ATP binding; growth hormone receptor binding; heme binding; histone binding; insulin receptor substrate binding; interleukin-12 receptor binding; metal ion binding; non-membrane spanning protein tyrosine kinase activity; peptide hormone receptor binding; phosphoinositide 3-kinase binding; protein binding; protein C-terminus binding; protein kinase activity; protein kinase binding; protein-tyrosine kinase activity; Ras guanyl-nucleotide exchange factor activity; receptor binding; SH2 domain binding; type 1 angiotensin receptor binding

Biological Process: actin filament polymerization; activation of MAPKK activity; adaptive immune response; apoptosis; axon regeneration; blood coagulation; caspase activation; cell differentiation; cell migration; cell motility; cytokine and chemokine mediated signaling pathway; elevation of cytosolic calcium ion concentration; enzyme linked receptor protein signaling pathway; erythrocyte differentiation; G-protein coupled receptor protein signaling pathway; induction of apoptosis by oxidative stress; innate immune response; JAK-STAT cascade; MAPKKK cascade; mesoderm development; mineralocorticoid receptor signaling pathway; negative regulation of cell proliferation; negative regulation of cell-cell adhesion; negative regulation of DNA binding; negative regulation of heart contraction; negative regulation of neuron apoptosis; peptidyl-tyrosine phosphorylation; platelet-derived growth factor receptor signaling pathway; positive regulation of cell activation; positive regulation of cell differentiation; positive regulation of cell migration; positive regulation of cell proliferation; positive regulation of DNA binding; positive regulation of GTPase activity; positive regulation of inflammatory response; positive regulation of insulin secretion; positive regulation of interleukin-1 beta production; positive regulation of nitric oxide biosynthetic process; positive regulation of nitric-oxide synthase biosynthetic process; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of phosphoprotein phosphatase activity; positive regulation of protein import into nucleus, translocation; positive regulation of transcription factor activity; positive regulation of tumor necrosis factor production; positive regulation of tyrosine phosphorylation of Stat3 protein; positive regulation of tyrosine phosphorylation of Stat5 protein; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of apoptosis; regulation of cell proliferation; regulation of inflammatory response; response to antibiotic; response to hydroperoxide; response to lipopolysaccharide; signal transduction; STAT protein nuclear translocation; tumor necrosis factor-mediated signaling pathway; tyrosine phosphorylation of JAK2 protein; tyrosine phosphorylation of STAT protein; tyrosine phosphorylation of Stat1 protein; tyrosine phosphorylation of Stat3 protein; tyrosine phosphorylation of Stat5 protein

Disease: Budd-chiari Syndrome; Erythrocytosis, Familial, 1; Myelofibrosis; Polycythemia Vera; Thrombocythemia 3

NCBI Summary:This gene product is a protein tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. It has been found to be constituitively associated with the prolactin receptor and is required for responses to gamma interferon. Mice that do not express an active protein for this gene exhibit embryonic lethality associated with the absence of definitive erythropoiesis. [provided by RefSeq, Jul 2008]
UniProt Code:O60674
NCBI GenInfo Identifier:12643404
NCBI Gene ID:3717
NCBI Accession:O60674.2
UniProt Secondary Accession:O60674,O14636, O75297,
UniProt Related Accession:O60674
Molecular Weight:130,674 Da
NCBI Full Name:Tyrosine-protein kinase JAK2
NCBI Synonym Full Names:Janus kinase 2
NCBI Official Symbol:JAK2
NCBI Official Synonym Symbols:JTK10; THCYT3
NCBI Protein Information:tyrosine-protein kinase JAK2
UniProt Protein Name:Tyrosine-protein kinase JAK2
UniProt Synonym Protein Names:Janus kinase 2; JAK-2
Protein Family:Tyrosine-protein kinase
UniProt Gene Name:JAK2
UniProt Entry Name:JAK2_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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