Human ANG-R-Tie2 (Angiopoietin Receptor Tie2) ELISA Kit (HUES01559)

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HUES01559
€539

Description

ELISA Kit Technical ManualMSDS

Human ANG-R-Tie2 (Angiopoietin Receptor Tie2) 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 ANG-R-Tie2 . 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 ANG-R-Tie2 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 ANG-R-Tie2, 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 ANG-R-Tie2. The concentration of Human ANG-R-Tie2 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 Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human ANG-R-Tie2 in samples. No significant cross-reactivity or interference between Human ANG-R-Tie2 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
(ng/mL)
O.D Average Corrected
102.528
2.54
2.5342.445
51.692
1.734
1.7131.624
2.50.923
0.923
0.9230.834
1.250.463
0.499
0.4810.392
0.630.285
0.269
0.2770.188
0.320.195
0.189
0.1920.103
0.160.142
0.142
0.1420.053
00.084
0.094
0.089--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ANG-R-Tie2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ANG-R-Tie2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.501.324.230.541.334.53
Standard deviation0.030.070.130.040.070.15
C V (%)6.005.303.077.415.263.31

Recovery

The recovery of Human ANG-R-Tie2 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)91-10397
EDTA plasma (n=5)90-10596
Cell culture media (n=5)95-108101

Linearity

Samples were spiked with high concentrations of Human ANG-R-Tie2 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 (%)91-10295-10893-109
Average (%)96101100
1:4Range (%)91-10580-9085-100
Average (%)988591
1:8Range (%)92-10783-9487-100
Average (%)998894
1:16Range (%)89-10384-9681-93
Average (%)958987

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 ANG-R-Tie2 (Angiopoietin Receptor Tie2) ELISA Kit (HUES01559) 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 ANG-R-Tie2 (Angiopoietin Receptor Tie2) ELISA Kit (HUES01559) Protein Information

UniProt Protein Function:TIE2: a receptor tyrosine kinase of the Tie family. Receptor for angiopoietin 1. Expressed almost exclusively in endothelial cells. May constitute the earliest mammalian endothelial cell lineage marker. Appears to be critical for endothelial cell-smooth muscle cell communication in venous morphogenesis. TEK is closely related to the TIE receptor tyrosine kinase.
UniProt Protein Details:

Protein type:Protein kinase, TK; Protein kinase, tyrosine (receptor); EC 2.7.10.1; Kinase, protein; Membrane protein, integral; TK group; Tie family

Chromosomal Location of Human Ortholog: 9p21

Cellular Component: microvillus; focal adhesion; cell surface; basolateral plasma membrane; integral to plasma membrane; extracellular region; actin filament; intercellular junction; lipid raft; perinuclear region of cytoplasm; apical plasma membrane; stress fiber; basal plasma membrane; plasma membrane

Molecular Function:protein binding; growth factor binding; protein-tyrosine kinase activity; receptor activity; transmembrane receptor protein tyrosine kinase activity; ATP binding; protein kinase activity

Biological Process: response to peptide hormone stimulus; peptidyl-tyrosine phosphorylation; response to cAMP; heart development; protein amino acid autophosphorylation; cell-matrix adhesion; positive regulation of cytokine secretion during immune response; signal transduction; cell-cell adhesion; cell-cell signaling; positive regulation of focal adhesion formation; angiogenesis; endochondral ossification; organ regeneration; Tie receptor signaling pathway; regulation of establishment and/or maintenance of cell polarity; positive regulation of phosphoinositide 3-kinase activity; positive regulation of peptidyl-serine phosphorylation; protein oligomerization; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of protein kinase B signaling cascade; negative regulation of angiogenesis; positive regulation of angiogenesis; positive regulation of protein import into nucleus; response to estrogen stimulus; negative regulation of inflammatory response; endothelial cell proliferation; response to hypoxia; positive regulation of endothelial cell proliferation; regulation of vascular permeability; sprouting angiogenesis; positive regulation of protein amino acid phosphorylation; blood coagulation; leukocyte migration; transmembrane receptor protein tyrosine kinase signaling pathway; negative regulation of apoptosis

Disease: Venous Malformations, Multiple Cutaneous And Mucosal

NCBI Summary:The TEK receptor tyrosine kinase is expressed almost exclusively in endothelial cells in mice, rats, and humans. This receptor possesses a unique extracellular domain containing 2 immunoglobulin-like loops separated by 3 epidermal growth factor-like repeats that are connected to 3 fibronectin type III-like repeats. The ligand for the receptor is angiopoietin-1. Defects in TEK are associated with inherited venous malformations; the TEK signaling pathway appears to be critical for endothelial cell-smooth muscle cell communication in venous morphogenesis. TEK is closely related to the TIE receptor tyrosine kinase. [provided by RefSeq, Jul 2008]
UniProt Code:Q02763
NCBI GenInfo Identifier:218511853
NCBI Gene ID:7010
NCBI Accession:Q02763.2
UniProt Secondary Accession:Q02763,Q5TCU2, Q8IV34, A8K6W0, B4DH20, B4DHD3, D3DRK5 E7EWI2,
UniProt Related Accession:Q02763
Molecular Weight:60.2kD
NCBI Full Name:Angiopoietin-1 receptor
NCBI Synonym Full Names:TEK tyrosine kinase, endothelial
NCBI Official Symbol:TEK  
NCBI Official Synonym Symbols:TIE2; VMCM; TIE-2; VMCM1; CD202B  
NCBI Protein Information:angiopoietin-1 receptor; hTIE2; p140 TEK; soluble TIE2 variant 1; soluble TIE2 variant 2; endothelial tyrosine kinase; tyrosine-protein kinase receptor TEK; tunica interna endothelial cell kinase; tyrosine-protein kinase receptor TIE-2; tyrosine kinase with Ig and EGF homology domains-2
UniProt Protein Name:Angiopoietin-1 receptor
UniProt Synonym Protein Names:Endothelial tyrosine kinase; Tunica interna endothelial cell kinase; Tyrosine kinase with Ig and EGF homology domains-2; Tyrosine-protein kinase receptor TEK; Tyrosine-protein kinase receptor TIE-2; hTIE2; p140 TEK
Protein Family:Angiopoietin-1 receptor
UniProt Gene Name:TEK  
UniProt Entry Name:TIE2_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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