Instruments for the investigation of protein-protein interactions

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.

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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

Method	Apollo 11 LB 913	Centro LB 963	Tristar 3	Tristar 5	Mithras² LB 943
Yeast Two Hybrid system
Split-ubiquitin system
FRET (SE FRET)
TR-FRET
BiFC
Fluorescence Lifetime FRET
Split-luciferase complementation
BRET

PPI techniques using fluorescence microscopes

Fluorescence microscopes (usually confocal ones) can be used to visualize the site of protein-protein interactions at the tissue and cell level. Some microscopes are very sophisticated instruments that can be expanded using hardware add-ons to be able to perform a broad variety of techniques useful in the study of PPI, such as Fluorescence Lifetime Imaging (FLIM). However, they have several constraints: they provide low throughput, because samples have to be examined one by one, and they are not suitable for luminescence-based methods. Further disadvantages include photobleaching and phototoxicity caused by the long exposure of the samples to light during observation (phenomena normally absent in microplate readers because of the short measurement times used, typically around 0,1 s). Fluorescence microscopes are most often used to perform FRET, FLIM FRET and BiFC. A detailed discussion about fluorescence microscopes is out of the scope of this article.

PPI techniques using In Vivo Imaging Systems

In Vivo Imaging Systems, such as fluorescence microscopes, also rely on imaging to localize and visualize protein-protein interactions, but this technology uses macroscopic samples (mice, rats, seedlings, leaves, plants in pots...). In vivo imaging systems are therefore the instrument of choice for localising the PPI within the organism, or for applying techniques that are unavailable with a microscope, such as luminescence-based methods. However, fluorescence sensitivity of in vivo imaging systems is significantly lower than that of fluorescence microscopes, which can make it difficult to detect FRET, for example, unless the light emission is strong and occurs on the surface of the sample (leaves, skin, ...). This is a consequence of less light reaching the sample per surface unit because large samples are used. The thickness of the sample also results in much higher light absorption and scattering of both, excitation and emission light. The throughput of in vivo imaging systems is higher than that of microscopes because the large image area allows several animals or plants to be imaged simultaneously.

In vivo imaging systems are a popular solution for performing split-luciferase complementation assays to detect protein-protein interactions in planta. This can be achieved simply by transforming leaves of the plant of interest, and the same leaf can be transformed with different combinations of the reporter system with the proteins of interest. The NightShade LB 985 is a popular system for this application. Good results for split-luciferase complementation assays have been published, for example, in Lin et al. 2019, Liu et al. 2017,Dong et al. 2018, and Yang et al. 2019.

In addition to the split-luciferase complementation assays, BRET and BiFC have been successfully performed using in vivo imaging systems. FRET has also been reported but, to our knowledge, not for the study of protein-protein interactions.

The following table summarizes the suitability of the different systems and the respective techniques for the investigation of in vivo protein-protein interactions:

Comparison of instruments for in vivo investigation of PPI

Method	Microplate Reader	Fluorescence Microscope	In Vivo Imaging System
Yeast Two Hybrid system
Split-ubiquitin system
FRET (SE FRET)			?
TR-FRET
BiFC			***
Fluorescence Lifetime FRET	*	**
Split-luciferase complementation
BRET			***
Throughput	+++	-	+
Localization	-	++	++
Fluorescence performance	+++	++	+
Luminescence performance	+++	-	++

* Only one system commercially available

** Expensive hardware add-ons needed

*** Very little data available; more testing needed

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