PPI techniques using microplate readers

Microplate Readers are a broad group of instruments that include both, single technology readers and multimode readers. The main advantage of microplate readers over other available instruments for measuring PPI techniques is that they can measure many samples in a relatively short time: all readers can measure 96-well microplates, and some can also measure 384-well and 1536-well microplates. In addition, they have excellent sensitivity for both, fluorescence and luminescence measurements due to the adoption of photomultiplier tubes that can detect even the smallest amounts of photons. However, compared to imaging instruments such as confocal microscopes and in vivo imaging systems, microplate readers provide virtually no spatial information that would allow localization of the interaction.

A simple absorbance microplate reader equipped with suitable filters is usually sufficient to quantify the activity of the chromogenic enzymes used in the yeast two-hybrid and split-ubiquitin systems. A monochromator-based absorption reader is also well suited for this application.

Any microplate luminometer or tube luminometer can be used to quantify the luminescence emitted during split-luciferase complementation assay. For BRET assays, however, multimode readers should be used as they can be equipped with the necessary filters to distinguish the acceptor emission from that of the donor. BRET assays can also be performed with monochromators; however, the sensitivity is much lower than when using filters and is therefore not recommended.

Both FRET  and Bimolecular Fluorescence Complementation (BiFC) can be measured using any fluorescence microplate reader with suitable filters. However, since the spectral range of the possible FRET pairs is very wide, it is advisable to test any fluorophore emitting at wavelengths greater than 600 nm against the spectral range of the instrument, as in some readers an extension of the spectral range to 850 or 900 nm is optional. As in the case of BRET, FRET performance when using filters is generally better than with monochromators.

Although it is common for modern microplate readers to be able to measure TR-FRET, it is sometimes an optional feature that may be missing in low-end or older instruments. However, the term TR-FRET is hardly used in the specifications: instead, look for TRF (Time-Resolved Fluorescence). In fact, from an instrument perspective a TR-FRET measurement is only a combination of 2 TRF measurements - one for the donor and one for the acceptor. TR-FRET measurements also have better performance when measured with filters instead of monochromators, and it has to be demonstrated again that the instrument's spectral range enables measurement of all fluorophores involved, since measurement at 665 nm is common in some TR-FRET assays.

Lifetime Fluorescence measurements require very specific hardware and are not available in any multimode reader. While several different instruments have been available in the past from major manufacturers, including Tecan and Berthold Detection Systems (now a part of Berthold Technologies), most of them have been discontinued and, currently, the only microplate reader available with Lifetime Fluorescence capabilities is the NovaFluor PR from Fluorescence Innovations.

Microplate Reader Recommendations from Berthold Technologies for PPI Studies

MethodApollo 11 LB 913Centro LB 960Tristar 3Tristar 5Mithras² LB 943

Yeast Two Hybrid system

     
Split-ubiquitin system     
FRET (SE FRET)     
TR-FRET     
BiFC     
Fluorescence Lifetime FRET     
Split-luciferase complementation     
BRET