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. They all have excellent sensitivity for both fluorescence and luminescence measurements due to the adoption of photomultiplier tubes because PMTs can detect even the smallest amounts of photons. But compared to imaging systems like 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 enough to quantify the activity of 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 or tube luminometer can be used to quantify the luminescence emitted during split-luciferase complementation assay. For BRET assaysmultimode readers should be used as they can be equipped with the necessary filters to distinguish the acceptor emission from the donor. BRET assays can also be performed with monochromators, but 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. 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 because 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). 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 must be demonstrated that the instruments 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 3

Yeast Two Hybrid system

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