Summary for Proteome Profiler Human Phospho-RTK Array Kit
Kit Summary
A membrane-based antibody array for the parallel determination of the relative phosphorylation of human receptor tyrosine kinases. Validated for analyte detection in cell lysates.
Detects phosphorylation of 49 human receptor tyrosine kinases simultaneously
Requires no specialized equipment
Principle of the Assay
The Proteome Profiler Human Phospho-RTK Array Kit is a membrane-based sandwich immunoassay. Capture antibodies spotted in duplicate on nitrocellulose membranes bind to specific target proteins present in the sample (Step 1). Tyrosine phosphorylation of the captured proteins is detected with an HRP-conjugated pan phospho-tyrosine antibody (Step 2) and then visualized using chemiluminescent detection reagents (Step 3). The signal produced is proportional to the amount phosphorylation in the bound analyte.
Why Use an Antibody Array to Detect Receptor Phosphorylation?
Determining the phosphorylation of multiple receptors in a single sample can be expensive, time consuming and can require specialized equipment. Performing multiple immunoprecipitations and Western blots requires time, labor, and reagents. The use of a multiplex antibody array to detect multiple phosphorylations in a single sample can be cost-effective and also save time and sample.
Kit Contents
4 Array Membranes
4-Well Multi-dish
Array Buffers
Lysis Buffer
Wash Buffer
Anti-Phospho-Tyrosine-HRP Detection Antibody
Chemiluminescent Detection Reagents
Transparency Overlay Template
Detailed Protocol
For a complete list of the kit contents and necessary materials, please see the Materials Provided/Other Supplies Required sections of the product datasheet.
Stability and Storage
Store the unopened kit at 2 °C to 8 °C. Do not use past kit expiration date.
Simultaneously detect the relative phosphorylation of these RTKs in a single sample
ALK/CD246
EphB4
MuSK
Axl
EphB6
PDGF R alpha
DDR1
ErbB2
PDGF R beta
DDR2
ErbB3
c-Ret
Dtk
ErbB4
ROR1
EGF R
FGF R1
ROR2
EphA1
FGF R2 alpha
Ryk
EphA2
FGF R3
SCF R/c-kit
EphA3
FGF R4
Tie-1
EphA4
Flt-3/Flk-2
Tie-2
EphA5
HGF R/c-MET
TrkA
EphA6
IGF-I R
TrkB
EphA7
Insulin R/CD220
TrkC
EphA10
M-CSF R
VEGF R1/Flt-1
EphB1
Mer
VEGF R2/KDR
EphB2
MSP R/Ron
VEGF R3/Flt-4
EphB3
Assays for Analytes represented in the Proteome Profiler Human Phospho-RTK Array Kit
Receptor Tyrosine Kinases (RTKs) are widely expressed transmembrane proteins that act as receptors for growth factors, neurotrophic factors, and other extracellular signaling molecules. Upon ligand binding, they undergo tyrosine phosphorylation at specific residues in the cytoplasmic tail. This leads to the binding of protein substrates and/or the establishment docking sites for adaptor proteins involved in RTK-mediated signal transduction. RTKs have critical functions in several developmental processes including regulating cell survival, proliferation, and motility. When unregulated, they play prominent roles in cancer formation. R&D Systems offers several different multiplex assay formats that allow the user to assess the activity of many RTKs in a single sample. Available RTK multiplex kits include bead-based multiplex kits for the Luminex platform, membrane-based antibody arrays, and microplate-based antibody arrays.
Product Specifications for Proteome Profiler Human Phospho-RTK Array Kit
Species
Human
Source
N/A
Scientific Data Examples for Proteome Profiler Human Phospho-RTK Array Kit
Detection of Human Receptor Tyrosine Kinase Phosphorylation in KATO-III Cell Line.
KATO-III human gastric carcinoma cells were either untreated or treated with 100 ng/mL rhFGF acidic and 1 μg/mL heparin for 15 minutes. Cells for rhFGF acidic/Heparin treatment either received pre-treatment with the reversible FGF R and VEGF R inhibitor PD 173074 (Tocris, Catalog # 3044), or with the FGF R-specific tyrosine kinase inhibitor SU 5402 (Tocris, Catalog # 3300), or no pre-treatment.
The Human Phospho-RTK Array is specific for ErbB2, ErbB3, and ErbB4 as shown by receptor competition.
MDA-MB-453 human breast cancer cells were treated with 100 ng/mL of rhNRG1-beta1/HRG1-beta1 (R&D Systems, Catalog # 396-HB) for 5 minutes. 5 µg of rhErbB2 (R&D Systems, Catalog # 1129-ER), rhErbB3 (R&D Systems, Catalog # 348-RB), or rhErbB4 (R&D Systems, Catalog # 1131-ER) extracellular domains were added to 50 µg of lysate and analyzed using the Human Phospho-RTK Array. Competition of a particular ErbB receptor was observed only with the corresponding recombinant soluble receptor.
Preparation & Storage
Shipping Conditions
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.