are a broad group of instruments that include both, single technology readers and . 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 , microplate readers provide virtually no spatial information that would allow localization of the interaction.
A simpleequipped 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.
Anyor can be used to quantify the luminescence emitted during split-luciferase complementation assay. For , however, 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 ; however, the sensitivity is much lower than when using filters and is therefore not recommended.
Bothand Bimolecular Fluorescence Complementation (BiFC) can be measured using any 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 .
Although it is common for modern microplate readers to be able to measure, 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 . 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.
Microplate Reader Recommendations from Berthold Technologies for PPI Studies
Yeast Two Hybrid system
|FRET (SE FRET)|
|Fluorescence Lifetime FRET|