Mouse Cell Signalling ELISA Kits 6

Mouse Tyrosine-protein kinase JAK2 (Jak2) ELISA Kit

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SKU:
MOEB2364

Description

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Product Name:Mouse Tyrosine-protein kinase JAK2 (Jak2) ELISA Kit
Product Code:MOEB2364
Alias:Tyrosine-protein kinase JAK2, Janus kinase 2, JAK-2, Jak2, 2.7.10.2
Uniprot:Q62120
Reactivity:Mouse
Range:0.312-20 ng/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
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.Protein type: Protein kinase, TK; Kinase, protein; Protein kinase, tyrosine (non-receptor); EC 2.7.10.2; Oncoprotein; TK group; JakA familyChromosomal Location of Human Ortholog: 9p24Cellular Component: nucleoplasm; extrinsic to internal side of plasma membrane; cytoskeleton; nuclear matrix; cytoplasm; caveola; nucleus; cytosol; lipid raftMolecular Function: protein C-terminus binding; histone binding; non-membrane spanning protein tyrosine kinase activity; acetylcholine receptor binding; protein kinase binding; protein kinase activity; interleukin-12 receptor binding; protein binding; growth hormone receptor binding; peptide hormone receptor binding; insulin receptor substrate binding; protein-tyrosine kinase activity; heme binding; phosphoinositide 3-kinase binding; type 1 angiotensin receptor binding; SH2 domain binding; ATP binding; receptor bindingBiological Process: establishment and/or maintenance of chromatin architecture; positive regulation of nitric oxide biosynthetic process; activation of MAPKK activity; tyrosine phosphorylation of Stat3 protein; response to lipopolysaccharide; tyrosine phosphorylation of JAK2 protein; protein amino acid phosphorylation; enzyme linked receptor protein signaling pathway; positive regulation of tyrosine phosphorylation of Stat3 protein; regulation of apoptosis; response to antibiotic; elevation of cytosolic calcium ion concentration; tumor necrosis factor-mediated signaling pathway; erythrocyte differentiation; mesoderm development; negative regulation of neuron apoptosis; positive regulation of cell activation; positive regulation of DNA binding; positive regulation of protein import into nucleus, translocation; axon regeneration; positive regulation of insulin secretion; JAK-STAT cascade; positive regulation of tumor necrosis factor production; tyrosine phosphorylation of Stat5 protein; positive regulation of phosphoinositide 3-kinase cascade; tyrosine phosphorylation of STAT protein; positive regulation of peptidyl-tyrosine phosphorylation; response to hydroperoxide; mineralocorticoid receptor signaling pathway; tyrosine phosphorylation of Stat1 protein; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription factor activity; positive regulation of cell differentiation; positive regulation of nitric-oxide synthase biosynthetic process; positive regulation of phosphoprotein phosphatase activity; peptidyl-tyrosine phosphorylation; hormone-mediated signaling; negative regulation of heart contraction; apoptosis; protein amino acid autophosphorylation; platelet-derived growth factor receptor signaling pathway; signal transduction; host programmed cell death induced by symbiont; negative regulation of cell proliferation; actin filament polymerization; positive regulation of cell proliferation; cell differentiation; STAT protein nuclear translocation; caspase activation; cell migration; cytokine and chemokine mediated signaling pathway; negative regulation of DNA binding; regulation of cell proliferation; positive regulation of interleukin-1 beta production; G-protein coupled receptor protein signaling pathway; positive regulation of tyrosine phosphorylation of Stat5 protein; regulation of inflammatory response; innate immune response; negative regulation of cell-cell adhesion; cell motility; blood coagulation; induction of apoptosis by oxidative stress; positive regulation of cell migration; positive regulation of inflammatory responseDisease: Polycythemia Vera; Myelofibrosis; Budd-chiari Syndrome; Erythrocytosis, Familial, 1; Thrombocythemia 3
UniProt Protein Details:
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:Q62120
NCBI GenInfo Identifier:4826776
NCBI Gene ID:3717
NCBI Accession:NP_004963.1
UniProt Secondary Accession:Q62120,Q62120, Q62689
UniProt Related Accession:Q62120,O60674
Molecular Weight:
NCBI Full Name:tyrosine-protein kinase JAK2 isoform a
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
Protein Family:Tyrosine-protein kinase
UniProt Gene Name:JAK2  
UniProt Entry Name:JAK2_HUMAN
Component Quantity (96 Assays) Storage
ELISA Microplate (Dismountable) 8×12 strips -20°C
Lyophilized Standard 2 -20°C
Sample Diluent 20ml -20°C
Assay Diluent A 10mL -20°C
Assay Diluent B 10mL -20°C
Detection Reagent A 120µL -20°C
Detection Reagent B 120µL -20°C
Wash Buffer 30mL 4°C
Substrate 10mL 4°C
Stop Solution 10mL 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

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1. Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2. Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3. Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4. Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5. Repeat the wash process for five times as conducted in step 3.
6. Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7. Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9. After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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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Additional Information

Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q62120
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Reactivity:
Mouse
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