TR-FRET

Kinases are enzymes that place a phosphate group on a serine/threonine or a tyrosine residue of a protein or peptide. All kinase reactions use ATP as the phosphate source. Therefore there have been assays developed that monitor the loss or gain of the peptide/protein substrate (LANCE, ULight) [23], the loss of ATP (easylite luminescence kinaseGlo, Perkin Elmer) [20], or the gain of ADP (Tran-screener TR-FRET) [24]. Many of these formats are applicable to cell based assays. 

There are cAMP assays that can be measured using a variety of techniques [35], including HTRF (CisBio) [36], bioluminescence (cAMP-Glo, Promega) [37], TR-FRET (CisBio) [38], LANCE [39], Alphascreen (Perkin Elmer) [40] and Enzyme Fragment Complementation (EFC) [41] (Hithunter, DiscoverX). This review will focus on the LANCE, the TR-FRET and ECF assays as they are the most used in our laboratories. 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Fig. 6 Assay formats for LANCE (left) and TR-FRET (right) for the detection of cAMP...

The truth most likely lies somewhere in between. Bender [67] published the most quantitative study to date on the success of HTS at Novartis. Several conclusions could be drawn. Particular target types and assay technologies have a great impact on screening success, and this was not always correlated to the number of identifying hits in the HTS runs. For assay formats used a minimum of five times, LC/MS readouts succeed 83% of the time, followed by FP assays, which succeed in 72% of the cases. TR-FRET showed a success rate of 70%, with FLIPR assays (61%), fluorescence intensity readouts (59%), and AlphaScreen (60%) performing... 

In order to determine whether compounds identified in the primary HTS screen are specific, a counterscreen is required to identify and eliminate false positives that will arise in the primary screen. For protein—protein interaction screens, it is preferable to test an unrelated protein pair that uses the same mode of detection. For our purposes, we adapted a previously described TR-FRET assay that monitors the interaction between bacterial Staphylococcus aureus Dnal and phage protein 77ORF104 (Liu et al., 2004). 

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

When used with europium or terbium ions, a carbostyril-based lanthanide chelate can be excited at 340 nm and provide sharp characteristic emission bands for transfer of energy to the appropriate acceptor fluor. Similar to the TMT chelator described previously, luminescence from terbium FRET signals well with Cy3 dyes and luminescence from europium can be used with APC or Cy5 dyes. Other fluorescent dyes that have similar excitation and emission ranges to these also can be used as acceptors in TR-FRET assays. For instance, terbium chelates can... 

Comley, J. (2006) TR-FRET based assays-getting better with age. Drug Discov. World 7(2), 22-38. 

HTRF (homogeneous time-resolved fluorescence) LANCE (lanthanide chelate excitation) Cisbio International PerkinElmer Life Sciences Lanthanide TR-FRET using Eu3+ cryptate/chelate-donor fluorophore and cross-linked aUophycocyanin-acceptor fluorophore... 

LanthaScreen Invitrogen Lanthanide TR-FRET using Tb3+ chelate-donor fluorophore and fluorescein-acceptor fluorophore... 

Scintillation proximity (FlashPlate, SPA) Product of reaction is a 33P labeled peptide biotin that can be captured on a detection bead that scintillates from proximity to 33P dephosphorylation by phosphatases can High-throughput, relatively artifact free in imaging-based systems universal readout for kinases versatile Radioactive waste disposal may be less sensitive than TR-FRET Park (1999) Sills (2002) von Ahsen (2006)... 

Antibody-dependent phosphopeptide detection ZTV 660nM TR-FRET,... 

The availability of anti-phosphotyrosine antibodies, anti-phosphopeptide antibodies, and antibodies to fluorescent ADP analogs enabled the performance of several homogeneous methods using fluorophores including TR-FRET and fluorescence polarization (FP). 

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