TR-FRET Assays - New from Biorbyt

Homogeneous TR-FRET based immunoassays for detection of phosphorylated or total proteins in cell lysates

Biorbyt TR-FRET Cellular Phosphorylation Detection Kits are designed for the detection of specific phosphorylated or total (phosphorylated and non-phosphorylated) proteins in cell lysate samples using a simple (add-and-read protocol), homogeneous (no wash steps) TR-FRET assay. These rapid, convenient and sensitive assays monitor pathway activation or inhibition in both adherent and suspension cells. These new assays represent a robust, reliable and rapid alternative to more conventional methods such as Western Blot, ELISA or expensive bead-based assays.

Features

  • Homogeneous, add-and-read assays; no wash steps

  • Ready-to-use kit: all required reagents included

  • More robust, convenient and faster than WB or ELISA

  • Site and signal pathway-specific

  • Works with many cell types

  • Compatible with most TR-FRET compatible plate readers

Applications

  • Cellular kinase assays

  • Receptor activation studies

  • Functional testing and screening of small compounds and biotherapeutics

  • Mechanism of action studies of small compounds and biotherapeutics

  • For basic research and drug discovery

Contents of kit (100 or 500 assay points)

  • Europium-labeled antibody

  • Acceptor-labeled antibody

  • Lysis buffer

  • Control cell lysate

  • Detection buffer

Complementary reagents

  • Control cell lysates: can be used in your experiments to ensure kit performance

  • Cell Lysis Buffers: five available lysis buffers that have been specifically designed to lyse cells under nondenaturing conditions and are fully compatible for TR-FRET applications

  • TR-FRET compatible Detection Buffer

Assay principle

TR-FRET

Biorbyt phosphorylation and total protein assays are sandwich immunoassays based on the homogeneous (no wash) TR-FRET (time-resolved fluorescence resonance energy transfer) technology. One antibody is labeled with a donor fluorophore (a Europium chelate) and the second antibody is labeled with an acceptor fluorophore (a small-molecule dye). Binding of antibodies to the target protein brings fluorophores into close proximity. Upon excitation at 320 or 340 nm, energy is transferred from the donor Europium chelate to the acceptor fluorophore. This results in the emission of light at 665 nm, which is proportional to the concentration of phosphorylated or total (both phosphorylated and unphosphorylated) protein in the cell lysate.

The assays can be run with both adherent and suspension cells. After cell treatment, lysis is conducted using the specific lysis buffer included in each kit. The lysis buffers of matched phosphorylated and total protein assays are compatible, enabling an analysis of both protein populations from the same lysate sample. All assays use the same protocol optimized for both half-area 96-well plates and low volume 384-well plates. Phosphorylated or total protein in the cell lysate is quantitatively detected in a single step using the TR-FRET assay kit reagents. No washing is needed at any step.

A two-plate protocol for most applications

All assays can be run under a two-plate assay protocol. Cells are plated, treated and then lysed in a 96 well culture plate. Lysates are then transferred to a 96 or 384 assay plate where the TR-FRET reagents are added for direct detection of phosphorylated or total protein. No wash steps are required.

A one-plate protocol for HTS applications

In this protocol suitable for HTS, plating, treating, lysing and detecting are all performed in a single 384 assay plate. No transfer or wash steps are required.

TR-FRET graph
TR-FRET graph
TR-FRET graph

Usage

This product is for RESEARCH USE ONLY. Not for diagnostic or therapeutic use.

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