Can the Human Phospho-RTK Array be used for measuring the relative levels of total RTKs present in a sample?

No. Although the array kit uses capture antibodies that recognize both phosphorylated and unphosphorylated RTKs, the detection antibody is a pan anti-phospho-tyrosine antibody which only detects phosphorylated tyrosines on activated RTKs.

To confirm positive signal for an RTK by IP-Western blot, are the capture antibodies from the Human Phospho-RTK Array Kit offered separately?

The identities of the capture antibodies on the Human Phospho-RTK Array are considered proprietary. Antibodies for each RTK analyte in the kit are available as well as Phospho-Tyrosine HRP-conjugated Antibody (Catalog #HAM1676). It can be challenging to directly compare Western Blot results of the linearized and reduced protein to Array detection of the native form. In ELISA, the protein will also be in the native form. ELISA kits are offered for many of the analytes targeted by the Array. These options are listed in a table on the Array product webpage.

What are the phosphorylation site(s) of the 49 different tyrosine residues on the RTKs?

This kit uses a pan anti-phospho-tyrosine antibody as the detection antibody, which means it is capable of detecting phosphorylation at any available tyrosine. It is not designed to be specific for one phosphorylation site on each molecule. For instance, PDGF R alpha has multiple sites of tyrosine phosphorylation (see the “PTM/Processing” section of http://www.uniprot.org/uniprot/P16234 for a list of the currently identified sites).

Which lysis buffers can be used for sample preparation?

Lysis Buffer 17 (Part #895943), provided in the kit, has been validated for optimal sample performance. Use of other lysis buffer requires independent evaluation.

Proteome Profiler Human Phospho-RTK Array Kit Best Seller

Name: Proteome Profiler Human Phospho-RTK Array Kit
Brand: R&D Systems
SKU: ARY001B
Rating: 5 (8 reviews)

R&D Systems, part of Bio-Techne | Catalog # ARY001B

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Product Datasheet / COA / SDS

Contains 4 membranes - each spotted in duplicate with 49 different RTK antibodies

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Product Summary
Scientific Data
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Species

Human

Product 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.

Troubleshooting Guide

Key Benefits

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

Analyte	Quantikine^® ELISA Kits	DuoSet^® ELISA Development System	DuoSet^® IC ELISA Development Systems (Total)	DuoSet^® IC ELISA Development Systems (Phospho)	Cell-based ELISA Kits
ALK/CD246
Axl		DY154	DYC1643
DDR1			DYC2396	DYC5859
DDR2			DYC2538	DYC6170
Dtk		DY859
EGF R	DEGFR0	DY231	DYC1854	DYC1095B
EphA1			DYC638
EphA2			DYC3035	DYC4056
EphA3
EphA4			DYC3038	DYC4057
EphA5
EphA6
EphA7
EphA10
EphB1
EphB2
EphB3
EphB4			DYC3038	DYC4057
ErbB2		DY1129	DYC1129	DYC1768
ErbB3		DY348	DYC234	DYC1769	KCB5677
ErbB4			DYC1133	DYC2115
ErbB6
FGF R1				DYC5079
FGF R2 alpha			DYC665	DYC684
FGF R3			DYC766	DYC2719
FGF R4			DYC685	DYC5516
Flt-3/Flk			DYC912	DYC368
HGF R/c-MET		DY358	DYC358	DYC2480
IGF-I R		DY391	DYC305	DYC1770
Insulin R/CD220			DYC1544	DYC2718
M-CSF R		DY329	DYC329	DYC3268
Mer		DY6488	DYC891	DYC2579
MSP R/Ron			DYC691	DYC1947
MuSK
PDGF R alpha			DYC322	DYC2114
Ret
ROR1
ROR2
Ryk
PDGF R beta			DYC385	DYC1767
PDGF R beta			DYC385	DYC3096
SCF R/c-kit	DSCR00	DY332	DYC332	DYC3527
Tie-1		DY5907
Tie-2	DTE200	DY5159		DCY2720
TrkA			DYC175	DYC2578
TrkB			DYC397	DYC688
TrkC			DYC373
VEGF R1/Flt-1	DVR100C	DY321B		DYC4170
VEGF R2/KDR	DVR200	DY357	DYC1780	DYC1766
VEGF R3/Flt-4		DY349	DYC3491	DYC2724

Scientific Data Images 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.

Formulation, Preparation, and Storage

Shipping

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.

Background: RTK Assay Kits

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.