Human ANG-R-Tie1 (Angiopoietin Receptor Tie1) ELISA Kit (HUES01558)

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HUES01558
€539

Description

ELISA Kit Technical ManualMSDS

Human ANG-R-Tie1 (Angiopoietin Receptor Tie1) 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-Tie1 . 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-Tie1 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-Tie1, 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-Tie1. The concentration of Human ANG-R-Tie1 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-Tie1 in samples. No significant cross-reactivity or interference between Human ANG-R-Tie1 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
102.442
2.458
2.452.393
51.601
1.661
1.6311.574
2.50.974
0.94
0.9570.9
1.250.456
0.462
0.4590.402
0.630.252
0.25
0.2510.194
0.320.164
0.154
0.1590.102
0.160.101
0.117
0.1090.052
00.057
0.057
0.057--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ANG-R-Tie1 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-Tie1 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.501.183.610.491.123.40
Standard deviation0.030.050.180.030.070.11
C V (%)6.004.244.996.126.253.24

Recovery

The recovery of Human ANG-R-Tie1 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)95-111102
EDTA plasma (n=5)93-10498
Cell culture media (n=5)89-9994

Linearity

Samples were spiked with high concentrations of Human ANG-R-Tie1 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 (%)96-10988-10293-107
Average (%)1019599
1:4Range (%)86-9881-9586-96
Average (%)928791
1:8Range (%)93-10686-9986-97
Average (%)999292
1:16Range (%)89-10081-9484-95
Average (%)958790

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-Tie1 (Angiopoietin Receptor Tie1) ELISA Kit (HUES01558) 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-Tie1 (Angiopoietin Receptor Tie1) ELISA Kit (HUES01558) Protein Information

UniProt Protein Function:TIE1: Transmembrane tyrosine-protein kinase that may modulate TEK/TIE2 activity and contribute to the regulation of angiogenesis. Belongs to the protein kinase superfamily. Tyr protein kinase family. Tie subfamily.
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 1p34-p33

Cellular Component: integral to plasma membrane

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

Biological Process: negative regulation of angiogenesis; peptidyl-tyrosine phosphorylation; response to retinoic acid; in utero embryonic development; mesoderm development; angiogenesis; signal transduction; vasculogenesis; negative regulation of cell migration; plasma membrane fusion

NCBI Summary:This gene encodes a member of the tyrosine protein kinase family. The encoded protein plays a critical role in angiogenesis and blood vessel stability by inhibiting angiopoietin 1 signaling through the endothelial receptor tyrosine kinase Tie2. Ectodomain cleavage of the encoded protein relieves inhibition of Tie2 and is mediated by multiple factors including vascular endothelial growth factor. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Nov 2011]
UniProt Code:P35590
NCBI GenInfo Identifier:4885631
NCBI Gene ID:7075
NCBI Accession:NP_005415.1
UniProt Related Accession:P35590
Molecular Weight:125,090 Da
NCBI Full Name:tyrosine-protein kinase receptor Tie-1 isoform 1
NCBI Synonym Full Names:tyrosine kinase with immunoglobulin-like and EGF-like domains 1
NCBI Official Symbol:TIE1  
NCBI Official Synonym Symbols:TIE; JTK14  
NCBI Protein Information:tyrosine-protein kinase receptor Tie-1; tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 1
UniProt Protein Name:Tyrosine-protein kinase receptor Tie-1
UniProt Gene Name:TIE1  
UniProt Entry Name:TIE1_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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