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Old technology, new application: the use of flow cytometry for high throughput analysis of protein-protein interactions

Figure 1. Schematic of the flow cytometry based Y2H array approach with liquid handling automation. Pictures of the instruments are from the vendors’ websites.
Figure 2. Flow cytometry analysis of GFP signal in 96-well plate. The GFP signal in each well at 0 hr and 72 hr time points was resolved by time. Nine wells containing the positive interaction control pair P53/T are marked in red.


by Jun Chen, Mark B. Carter, Bruce S. Edwards and Larry A. Sklar


The Y2H system has been the most commonly used method for large-scale analysis of protein-protein interactions. While the current Y2H array approach has been automated, it remains difficult due to intricate colony handling requirements. Here we describe an integrated Y2H array approach utilising liquid handling automation and employing high throughput flow cytometry analysis of the yEGFP reporter.


Y2H system

The identification and characterisation of protein-protein interactions (PPIs) is a key focus in post-genome research. The yeast two-hybrid system (Y2H) is the most commonly used method for large-scale identification of PPIs, and the experimental Y2H data play crucial roles in establishing large protein-protein networks (Interactomes) in various species including virus, bacterium, yeast, fly, worm and human. The Y2H system, developed two decades ago [1], is a genetic screen wherein the interaction between proteins of interest is detected by the reconstitution of a functional transcription factor, such as yeast Gal4p. Two proteins, one fused with the DNA binding domain (often called "Bait" or BD-X), the other fused with the transcriptional activation domain (often called "Prey" or AD-Y), are co-expressed in yeast cells. The interaction between the bait and prey proteins brings the DNA binding domain and the transcriptional activation domain into close proximity, forming a functional transcription factor which subsequently activates reporter gene expression. Typical Y2H assays aimed at fishing for PPIs in cDNA libraries, i.e. library screening approaches, have limited applications in the current high-throughput, large-scale Y2H campaign due to the tedious procedure and the cost of DNA sequencing for clone identification. Instead, the Y2H array (or matrix) approach in conjunction with increased access to automation has been employed for large-scale analysis. The array approach requires cloning of all individual cDNAs into the Y2H bait and prey vectors and comprehensively testing all possible bait/prey combinations. A one-to-one test of a binary protein interaction (the so-called matrix approach) is likely to be the most comprehensive and sensitive approach, but it requires a large number of time-consuming and labour-intensive manipulations for large sets of bait and prey. While pooling and mass-mating strategies have been developed to reduce labour, automation remains the key for performing large-scale Y2H assays.


Automation in Y2H

The current standard Y2H array approach is performed on agar plates by mating haploid cells on rich media plates, transferring colonies to the selective plates and scoring the positive PPIs on the selective plates [2]. While this procedure has been automated, it remains difficult because of the requirement for colony transfer. To eliminate the colony handling steps, the Y2H array approach was developed in liquid media by quantitative analysis of the LacZ reporter [3]. However, this procedure has been rarely used due to the requirement for lysing cells and adding substrates for measuring the LacZ reporter. Therefore, there remains a need to develop a novel, more efficient liquid handling automation procedure for such a Y2H array approach.


Flow cytometry and high throughput application

Modern flow cytometry has been a versatile high speed cell analysis method that has played key roles in proteomics and systems biology. The recent invention of the HyperCyt high throughput flow cytometry (HTFC), enables the processing of a 96- or 384-well plate in as little as 3 or 12 min, respectively [4]. This technology allows for the first time, the application of flow cytometry in the field of high throughput screening. To explore the utility of HTFC for large-scale analysis of PPIs, we sought to develop a flow cytometry-based Y2H system incorporating automation with liquid handling instruments.


GFPY2H system

To enable flow cytometry-based detection, we established a GFPY2H system wherein the yEGFP reporter was integrated into the chromosome of both mating types (a and α) of the yeast host strains (AH109 and Y187) in the Matchmaker Y2H system (Clontech) [5]. The green fluorescent signal triggered by PPIs in both host strains can be quantified in live yeast cells by flow cytometry without substrate addition or further treatment. The sensitivity of the yEGFP reporter is comparable to the classical nutrient or colorimetric reporters and allows detection of weak interactions. In addition, the quantitative analysis of GFP expression may allow identification of the binding partner of self-activators, such as eukaryotic transcription factors, which is difficult to achieve with the classical Y2H system. The GFPY2H affords advantages of rapid, convenient, sensitive and quantitative analysis of PPIs.


Integrated liquid handling automation procedure for Y2H array approach

We designed an integrated Y2H array approach that can be easily automated with liquid handling instruments: 1) mating bait and prey cells in liquid YPD media; 2) selecting diploid cells in selective media;and 3) subsequently analysing PPIs by flow cytometry. The major liquid handling instruments that are recommended for each step are listed in the right panel [Figure 1]. The mating step can be carried out with an automated pintool transfer instrument and a microplate dispenser. The most difficult step for automation is to change media after mating. A microplate washer can be applied to fulfill this task, but the yeast cells have to be spun down prior to liquid aspiration, a step that would benefit from the use of an automated centrifuge. After growth in the selective media for 3-4 days, the yeast cells are ready for analysis of GFP signal by the HyperCyt HTFC platform with the capability of processing ~120 96-well plates/day/cytometer. We have validated this approach using the control protein pairs in the Matchmaker Y2H system [6]. Briefly, we carried out a one-to-one test of 12 bait/prey combinations with 6-9 replicates for each pair in 96-well plates. A distinct population of GFP positive cells can be detected in the wells containing the positive interaction control pair P53/T by flow cytometry [Figure 2]. The key to successful application of this approach is to ensure that the diploid cells containing positive interaction pairs can overgrow in the culture within 3-4 days. This is dependent on the mating efficiency and the doubling time of the diploid cells. Since the doubling time is intricately determined by the protein pairs and often varies from 3-5 hrs, the mating efficiency becomes the key optimisable factor. In principle, as long as the mating procedure results in one diploid cell in the post-mating culture containing 106 cells, the diploid cell can reach 50% after 72-96 hrs growth, allowing robust detection of GFP signal by flow cytometry. Only a complete failure in the mating step will result in false negatives. It is unlikely a failure in mating will happen in a one-to-one test because a mating efficiency of 0.1% is routinely achievable in microplates. With a high complexity pooling and mass-mating strategy, such as 100 bait/prey matings in a single well, the percentage of the diploid cells containing each individual bait/prey pair in the post-mating culture is 100-fold lower than that in the one-to-one test. In this scenario, a mating efficiency that is lower than 0.1% may result in unsuccessful mating of some bait/prey pairs. Therefore, an optimised mating protocol is important and we do not recommend a pooling strategy that results in a mass-mating of greater than 100 bait/prey pairs in a single well. It is noteworthy that the multiplexing capability of flow cytometry is able to analyse multiple bait proteins simultaneously, allowing analysis of a "bait pool" in a mode of one-to-one tests of bait/prey combinations. This may increase the assay sensitivity for the pooling strategy without compromising the pooling complexity and the assay throughput. The pairing of the GFPY2H system with automated liquid handling is therefore a union of techniques that simplifies the handling and transfer of yeast colonies, gaining the advantages of speed and consistency from automated liquid handling with the speed and robust signal of flow cytometry.


Future prospects

This flow cytometry based Y2H array approach affords the advantages of automated liquid handling and quantitative reporter analysis, as well as detection of self-activators. It shows promise for future large-scale analysis of PPIs and adds a powerful new assay to the toolbox of flow cytometry-associated proteomics technologies. With further optimisation and miniaturisation, this Y2H array approach could become a useful tool in the large-scale analysis of PPIs, particularly, in the identification of host-pathogen PPIs.



1. Fields S and Song O Nature 1989; 340: 245-246.

2. Rajagopala SV and Uetz P Methods Mol Biol 2009; 548: 223-245.

3. Buckholz RG et al. J Mol Microbiol Biotechnol 1999; 1: 135-140.

4. Sklar LA et al. Curr Opin Pharmacol 2007; 7: 527-534.

5. Chen J et al. Cytometry A 2008; 73: 312-320.

6. Chen J et al. Cytometry A 2012; 81: 90-98.



This is supported by NIH U54 MH084690. BSE and LAS are inventors of HyperCyt and co-founders of IntelliCyt.


The authors

Jun Chen, Mark B. Carter, Bruce S. Edwards and Larry A. Sklar

UNM Center for Molecular Discovery,

Health Science Center,

University of New Mexico,

Albuquerque, NM 87131


Correspondence to Jun Chen or Larry A. Sklar



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