BACKGROUND
most if not all biological processes require the cooperation of pairs of proteins or the formation of large functional complexes of proteins.
therefore the analysis of protein-protein interactions, either in vitro, using for example protein arrays, co-immunoprecipitation or affinity chromatography, or in vivo by two-hybrid assays is essential for the elucidation of biological processes and/or networks.
in classical yeast or mammalian two-hybrid based assays typically two proteins of interest are ectopically expressed as fusion proteins, one with the dna binding domain  of for example gal <dig> or lexa and the other with a transcriptional activating domain , such that if both proteins show any interaction, the dbd and ad are functionally linked together at the promoter, reconstituting transcriptional activity  <cit> .
this causes reporters that contain gal4- or lexa binding sequences to be transcribed.
since two-hybrid systems are in vivo assays they offer advantages over in vitro biophysical or biochemical methods.
indeed some protein-protein interactions are too weak and/or transient to be detected in vitro and some of these interactions require specific post-translational modifications of the proteins and/or specific co-factors in the cellular microenvironment.
for the same reasons it is advantageous to determine protein interaction networks in mammalian cells, using mammalian two-hybrid assays  <cit> .
until now high-throughput analyses of mammalian protein interactions were typically performed in yeast  <cit>  and putative interactions were then confirmed in mammalian two-hybrid assays on a gene-by-gene scale  <cit> .
we present here a novel assay for the parallel analysis of thousands of proteins for interacting partners in mammalian cells by combining cell arrays  <cit> , with the more classical mammalian two-hybrid assay.
in this cell array protein-protein interaction assay , nanoliter volumes of solutions containing a bait expression plasmid, a prey expression plasmid and a reporter plasmid complexed with transfection reagent are immobilized on glass slides in well-defined array formats.
when these slides are overlayed with a monolayer of living cells, only those cells that grow on top of a particular spot of dna will get transfected and will start to over-express specific chimeric bait and prey proteins.
if these two proteins can interact with each other they will transactivate the autofluorescent reporter making that cluster of cells fluorescent while the surrounding cells remain non-fluorescent.
fluorescent cell clusters/features can then be analysed by simple fluorescence detection using common dna array scanners or high-throughput microscopy, without the need for further manipulation of the slides such as immunofluorescence staining or enzyme-based detection.
using known interacting proteins we demonstrate the specific and quantitative detection of protein-protein interactions on cell arrays in different mammalian cell lines.
furthermore, screening of  <dig> different combinations of prey and bait proteins including different domains of the human androgen receptor reveals that this assay is well suited for the detection of hormone-dependent protein interactions.
the physiological significance of this interaction on cell arrays is further underscored by showing the dose response of this interaction to androgenic compounds as well as to antagonistic reagents.
finally we demonstrate that the possible combinatorial screens to detect protein interactions can be increased by combining slides that only carry a prey library with cell lines that carry a stable or transient bait construct.
this flexibility, together with the high capacity of the cell arrays and the small amounts of reagents that are required, makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.
RESULTS
development of a cell array based method for the detection of protein-protein interactions in various mammalian cells
because cell arrays allow the simultaneous transfection and analysis of high numbers of different cdna constructs in adherent mammalian cells  <cit>  they offer the possibility to analyse thousands of proteins for interacting partners when combined with the mammalian two-hybrid assay.
to improve the ease and speed of detection of protein-protein interactions  on cell arrays we created an autofluorescence-based and gal4-driven reporter plasmid, gal4-pzsgreen.
similarly to other two-hybrid systems, expression of this reporter gene will only occur if two chimeric proteins, a bait fused to the gal <dig> dna-binding domain and a prey fused to an activation domain, are co-expressed and subsequently form a complex with transcriptional activity.
fluorescence detection is performed using common dna array scanners or high-throughput microscopy, eliminating the need for further manipulation of the slides.
this is demonstrated in figure  <dig>  where microarrays were made by printing the gal4-pzsgreen reporter mixed with pbd-p <dig> and pad-sv40t, coding for two interacting proteins or mixed with pbd-p <dig> and pad-traf, coding for two non-interacting proteins.
figure 1a, shows example images of different adherent cell lines transfected using identical microarray slides.
the resulting fluorescent signals, shown in figure 1b, were quantified as described in "methods".
using these plasmids we show the efficient transfection and quantitative detection of ppi in six phenotypically different cell lines.
interestingly, sample and slide preparation were the same for all cell lines tested and only the number of cells and time of transfection had to be optimized for each cell type.
the observation that specific protein-protein interactions can readily be analyzed in different cell lines is of particular interest for interactions that are dependent on cell-specific posttranslational modifications of expressed proteins or that depend on cell-specific co-factors.
a cell type dependent difference in ppi-induced reporter expression is already evident for p <dig> and sv40t proteins that induced higher reporter levels when expressed in pc- <dig> as compared to hek293t cells, even though both cell lines can be transfected with similar efficiencies, as reflected by the level of nf-κb-induced reporter expression in these cell lines.
application of cappia for screening of hormone-dependent protein interactions
having demonstrated the specificity and efficiency of cappia, we next explored whether this method allows the large scale screening for hormone-dependent interactions.
steroid hormone receptors such as the androgen receptor  are ligand-dependent transcription factors that control a variety of essential physiologic and developmental processes in humans  <cit> .
here, in the presence of  <dig> nm of the synthetic androgenic ligand r <dig>  we analysed the interaction of  <dig> individual baits with  <dig> different preys coding for proteins or protein domains potentially associated with nuclear receptor function .
each slide contained all  <dig> different prey-bait combinations as single features in addition to a series of positive and negative controls to localise the array and monitor transfection efficiency as well as background fluorescence.
in total each slide contained  <dig> spots, and three identical slides were transfected to obtain independent replicate data points for each prey-bait combination.
fluorescence signals of all  <dig> features were collected and processed as described in methods.
the  <dig> data points representing reporter expression induced by the different prey-bait combinations are summarized in figure  <dig>  bait b <dig> can immediately be recognized as being a so-called false positive bait since it activates reporter expression with multiple unrelated preys.
such false positives induce reporter expression either by unspecific binding or, as is the case for the fusion protein  of bait b <dig>  by conferring trans-activating activity to the gal <dig> dna binding domain of the bait  <cit> .
more importantly, a specific and androgen-dependent interaction was identified between bait b <dig>  the ligand binding domain of the ar , and prey p <dig>  the n-terminal domain of the ar .
this interaction was confirmed in a second cappia experiment .
these data are consistent with previously published androgen-dependent intra-molecular interactions between ar-lbd and ar-ntd domains  <cit> .
to evaluate the resolution of cappia and to obtain additional evidence for the physiological significance of the ar-lbd and ar-ntd interaction using cell arrays, a dose-response curve was determined.
we observed a dose-dependent induction of reporter expression in the presence of the synthetic agonist r <dig> with a maximal response from  <dig> nm onwards , in accordance with previous assays involving normal transfection of both domains and detection of gal4-induced luciferase activity  <cit> .
in addition we could reiterate the dose dependent inhibitory effects of two antagonists, medroxyprogesterone acetate  and hydroxyflutamide  on r1881-induced ar-lbd and ar-ntd interaction on cell arrays .
importantly the resolution of cappia allowed the detection of quantitative differences in antagonist activity.
indeed, the r1881-induced interaction was inhibited with  <dig> nm mpa, whereas almost  <dig> nm oh-flu was required to achieve the same level of inhibition.
similar differences in inhibitory potency between mpa and oh-flu were also observed after normal transient transfection of both ar domains in cho cells  <cit> .
taken together these experiments clearly demonstrate that cell arrays provide a functional readout to identify and monitor ppi under different physiological conditions.
besides interrogating ppi in different cellular contexts, cell arrays can also be used to screen for ligand-dependent protein-protein interactions and to quantify the dose response of these to various compounds.
pr-stable bait and pr-trans bait cell arrays
in order to increase the possible combinatorial screens for protein interactions and hence further improve the high-throughput application of cappia, we printed alternative slides on which the bait was omitted.
each spot on these so-called prey-reporter-  slides contains only the reporter and one of the prey constructs.
to screen for interacting partners the bait is then introduced into the cells before adding them to the prey-reporter-arrays.
this is either done using stably transfected cell lines  or by transiently transfecting cells with the bait shortly before adding them to the arrays .
in both cases all the cells on these transfected slides express the bait.
however, only cells that grow on top of a spot, with a prey that can interact with that bait, become fluorescent.
we used the hormone-dependent ar-lbd and ar-ntd interaction to compare the "pr-stable-bait" and "pr-trans-bait" strategies with the results obtained with the original prb slides described earlier.
a schematic representation of the three strategies with the corresponding results is shown in figure  <dig>  after normalisation of the data so as to correct for differences in transfection efficiencies the results show that all three strategies result in a comparable trans-activation of gal4-pzsgreen as a result of ar-lbd and ar-ntd interaction.
although prb slides offer a cost-effective and robust platform for simultaneous comparison of large number of interactions in different cell lines and under different culture conditions , the pr slides are more suitable for the large-scale screening of novel bait-prey interactions.
as shown in additional file  <dig>  the spot to spot distance on cappia slides can easily be reduced to  <dig> mm.
by printing each sample as one feature per slide and using a spot distance of  <dig> mm, up to  <dig> different preys can be printed on one slide.
replicate pr-slides can then be screened with any bait of interest, further increasing the high-throughput application of cappia.
in addition pr slides can be printed in large batches and can be stored for at least  <dig> months at 4°c and even longer at -80°c , thus increasing the cost effectiveness of cappia.
the slides can be shipped on ice, allowing any research group with access to standard culture facilities to interrogate the prey library with their bait of interest.
discussion
we hereby present a new screening technology for the high-throughput interrogation of protein-protein interactions in mammalian cells.
this cell-array-protein-protein-interaction-assay  combines the advantages of mammalian two-hybrid systems with those of microarrays, a combination that is expected to save considerable time and expense.
indeed, cappia slides are printed with the same robotic microarray devices used to print conventional dna microarrays.
consequently, cell arrays require far less dna, transfection reagents and cells as compared to assays performed in microwell plate format.
for comparison, the amount of transfection reagent required to transfect one sample in a  <dig> micro-well is sufficient to transfect more than  <dig> samples in cappia, a significant cost reduction since transfection reagents account for a major part of the expenses in these assays.
although cell arrays can theoretically carry up to  <dig> features per slide  <cit> , the actual number of features per slide is dependent on the type of application.
since the efficiency of the simultaneous transfection of three plasmids is lower than that of a single plasmid and since the reporter will only be expressed after sufficient amounts of both prey and bait proteins are expressed in the same cell there is a need to increase the number of cells per spot in cappia in order to guarantee the robust detection of interacting proteins.
consequently, in order to increase the sensitivity of the assay, the features on cappia slides need to be bigger and hence the slide capacity will be lower.
still, cappia slides can be printed with each containing up to  <dig> different preys as single spots when using a spot to spot distance of  <dig> mm.
this is equivalent to  <dig> standard  <dig> micro-well plates and data can then be collected from a number of identical slides to obtain replicate data points per sample.
in addition, by pooling  <dig> preys per feature even more preys can be interrogated per slide.
the cost-effectiveness of the assay is further increased by using an auto-fluorescent based reporter eliminating the need for immuno-fluorescence staining or enzyme-based reporter detection.
in contrast to microwell-based assays no extensive liquid handling infrastructure is required once the slides have been printed.
in addition pr or prb slides can be printed in large batches and stored frozen without losing their efficiency, thus increasing the flexibility and cost effectiveness of cappia.
finally, pr slides can be screened with any bait of interest simply by using standard transfection and cell culture methods, available to many research groups.
compared to the more common high-throughput yeast two-hybrid assay, cappia offers the advantage of testing mammalian ppi within a cellular context that more closely mimics the native protein environment.
as a consequence the rate of false negative interactions, i.e.
true interactions that can not be detected due to the inappropriate folding and/or post-translational processing of the proteins should be lower using cappia, especially since phenotypically different cell types can be used.
as with dna microarrays, pr-slides interrogated with different baits allow a comparison between multiple identical assays.
as such preys or baits that are found multiple times with unrelated proteins, so-called bait- or prey-unspecific false positives  <cit>  can be distinguished without need for further experimentation.
high-throughput yeast two-hybrid assays also require extensive liquid handling infrastructures  <cit>  making the assays less accessible for many research groups.
previously, suzuki et al  <cit>  described a pcr-mediated rapid sample preparation and a high-throughput assay system based on the mammalian two-hybrid method.
although the method they designed allows for the rapid preparation of high numbers of bait and prey samples, the actual two-hybrid assays are performed in microwells and require semiautomatic multiple dispensers as well as multiple reagents for downstream enzymatic detection of interacting proteins.
cappia, on the other hand allows the quantitative analysis of interactions comparable to assays performed in microwells, while offering the advantage of being economical and accessible to research groups equipped with basic cell culture infrastructures.
currently, large bait-prey libraries for use in cappia are being generated using gateway  compatible destination pcmv-ad and pcmv-bd vectors and panels of pentry clones.
since cappia is based on the gal4-two-hybrid system that is already used by many research groups, existing baits and preys can easily be included in the assays.
in particular, the trans-bait version of cappia readily allows available bait constructs to be screened against large number of preys.
CONCLUSIONS
taken together, the high capacity of the cell arrays, the flexibility to interrogate any bait of interest and the small amounts of reagents that are required make cappia currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.
microarray-based systems such as the one presented here should lead to the acceleration of the deciphering of functional protein complexes.
in addition cappia could also be an economical alternative for the identification of potential drug targets by combining screens for small molecule ligands that can disrupt or modulate interactions with selected prey-bait combinations expressed in array format inside  <dig> micro-wells  <cit> .

 most if not all biological processes require the cooperation of pairs of proteins or the formation of large functional complexes of proteins. therefore the analysis of protein-protein interactions, either in vitro, using for example protein arrays, co-immunoprecipitation or affinity chromatography, or in vivo by two-hybrid assays is essential for the elucidation of biological processes and/or networks. in classical yeast or mammalian two-hybrid based assays typically two proteins of interest are ectopically expressed as fusion proteins, one with the dna binding domain of for example gal or lexa and the other with a transcriptional activating domain, such that if both proteins show any interaction, the dbd and ad are functionally linked together at the promoter, reconstituting transcriptional activity. this causes reporters that contain gal4- or lexa binding sequences to be transcribed. since two-hybrid systems are in vivo assays they offer advantages over in vitro biophysical or biochemical methods. indeed some protein-protein interactions are too weak and/or transient to be detected in vitro and some of these interactions require specific post-translational modifications of the proteins and/or specific co-factors in the cellular microenvironment. for the same reasons it is advantageous to determine protein interaction networks in mammalian cells, using mammalian two-hybrid assays. until now high-throughput analyses of mammalian protein interactions were typically performed in yeast and putative interactions were then confirmed in mammalian two-hybrid assays on a gene-by-gene scale. we present here a novel assay for the parallel analysis of thousands of proteins for interacting partners in mammalian cells by combining cell arrays, with the more classical mammalian two-hybrid assay. in this cell array protein-protein interaction assay, nanoliter volumes of solutions containing a bait expression plasmid, a prey expression plasmid and a reporter plasmid complexed with transfection reagent are immobilized on glass slides in well-defined array formats. when these slides are overlayed with a monolayer of living cells, only those cells that grow on top of a particular spot of dna will get transfected and will start to over-express specific chimeric bait and prey proteins. if these two proteins can interact with each other they will transactivate the autofluorescent reporter making that cluster of cells fluorescent while the surrounding cells remain non-fluorescent. fluorescent cell clusters/features can then be analysed by simple fluorescence detection using common dna array scanners or high-throughput microscopy, without the need for further manipulation of the slides such as immunofluorescence staining or enzyme-based detection. using known interacting proteins we demonstrate the specific and quantitative detection of protein-protein interactions on cell arrays in different mammalian cell lines. furthermore, screening of different combinations of prey and bait proteins including different domains of the human androgen receptor reveals that this assay is well suited for the detection of hormone-dependent protein interactions. the physiological significance of this interaction on cell arrays is further underscored by showing the dose response of this interaction to androgenic compounds as well as to antagonistic reagents. finally we demonstrate that the possible combinatorial screens to detect protein interactions can be increased by combining slides that only carry a prey library with cell lines that carry a stable or transient bait construct. this flexibility, together with the high capacity of the cell arrays and the small amounts of reagents that are required, makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.  development of a cell array based method for the detection of protein-protein interactions in various mammalian cells because cell arrays allow the simultaneous transfection and analysis of high numbers of different cdna constructs in adherent mammalian cells they offer the possibility to analyse thousands of proteins for interacting partners when combined with the mammalian two-hybrid assay. to improve the ease and speed of detection of protein-protein interactions on cell arrays we created an autofluorescence-based and gal4-driven reporter plasmid, gal4-pzsgreen. similarly to other two-hybrid systems, expression of this reporter gene will only occur if two chimeric proteins, a bait fused to the gal dna-binding domain and a prey fused to an activation domain, are co-expressed and subsequently form a complex with transcriptional activity. fluorescence detection is performed using common dna array scanners or high-throughput microscopy, eliminating the need for further manipulation of the slides. this is demonstrated in figure where microarrays were made by printing the gal4-pzsgreen reporter mixed with pbd-p and pad-sv40t, coding for two interacting proteins or mixed with pbd-p and pad-traf, coding for two non-interacting proteins. figure 1a, shows example images of different adherent cell lines transfected using identical microarray slides. the resulting fluorescent signals, shown in figure 1b, were quantified as described in "methods". using these plasmids we show the efficient transfection and quantitative detection of ppi in six phenotypically different cell lines. interestingly, sample and slide preparation were the same for all cell lines tested and only the number of cells and time of transfection had to be optimized for each cell type. the observation that specific protein-protein interactions can readily be analyzed in different cell lines is of particular interest for interactions that are dependent on cell-specific posttranslational modifications of expressed proteins or that depend on cell-specific co-factors. a cell type dependent difference in ppi-induced reporter expression is already evident for p and sv40t proteins that induced higher reporter levels when expressed in pc- as compared to hek293t cells, even though both cell lines can be transfected with similar efficiencies, as reflected by the level of nf-κb-induced reporter expression in these cell lines. application of cappia for screening of hormone-dependent protein interactions having demonstrated the specificity and efficiency of cappia, we next explored whether this method allows the large scale screening for hormone-dependent interactions. steroid hormone receptors such as the androgen receptor are ligand-dependent transcription factors that control a variety of essential physiologic and developmental processes in humans. here, in the presence of nm of the synthetic androgenic ligand r we analysed the interaction of individual baits with different preys coding for proteins or protein domains potentially associated with nuclear receptor function. each slide contained all different prey-bait combinations as single features in addition to a series of positive and negative controls to localise the array and monitor transfection efficiency as well as background fluorescence. in total each slide contained spots, and three identical slides were transfected to obtain independent replicate data points for each prey-bait combination. fluorescence signals of all features were collected and processed as described in methods. the data points representing reporter expression induced by the different prey-bait combinations are summarized in figure bait b can immediately be recognized as being a so-called false positive bait since it activates reporter expression with multiple unrelated preys. such false positives induce reporter expression either by unspecific binding or, as is the case for the fusion protein of bait b by conferring trans-activating activity to the gal dna binding domain of the bait. more importantly, a specific and androgen-dependent interaction was identified between bait b the ligand binding domain of the ar, and prey p the n-terminal domain of the ar. this interaction was confirmed in a second cappia experiment. these data are consistent with previously published androgen-dependent intra-molecular interactions between ar-lbd and ar-ntd domains. to evaluate the resolution of cappia and to obtain additional evidence for the physiological significance of the ar-lbd and ar-ntd interaction using cell arrays, a dose-response curve was determined. we observed a dose-dependent induction of reporter expression in the presence of the synthetic agonist r with a maximal response from nm onwards, in accordance with previous assays involving normal transfection of both domains and detection of gal4-induced luciferase activity. in addition we could reiterate the dose dependent inhibitory effects of two antagonists, medroxyprogesterone acetate and hydroxyflutamide on r1881-induced ar-lbd and ar-ntd interaction on cell arrays. importantly the resolution of cappia allowed the detection of quantitative differences in antagonist activity. indeed, the r1881-induced interaction was inhibited with nm mpa, whereas almost nm oh-flu was required to achieve the same level of inhibition. similar differences in inhibitory potency between mpa and oh-flu were also observed after normal transient transfection of both ar domains in cho cells. taken together these experiments clearly demonstrate that cell arrays provide a functional readout to identify and monitor ppi under different physiological conditions. besides interrogating ppi in different cellular contexts, cell arrays can also be used to screen for ligand-dependent protein-protein interactions and to quantify the dose response of these to various compounds. pr-stable bait and pr-trans bait cell arrays in order to increase the possible combinatorial screens for protein interactions and hence further improve the high-throughput application of cappia, we printed alternative slides on which the bait was omitted. each spot on these so-called prey-reporter- slides contains only the reporter and one of the prey constructs. to screen for interacting partners the bait is then introduced into the cells before adding them to the prey-reporter-arrays. this is either done using stably transfected cell lines or by transiently transfecting cells with the bait shortly before adding them to the arrays. in both cases all the cells on these transfected slides express the bait. however, only cells that grow on top of a spot, with a prey that can interact with that bait, become fluorescent. we used the hormone-dependent ar-lbd and ar-ntd interaction to compare the "pr-stable-bait" and "pr-trans-bait" strategies with the results obtained with the original prb slides described earlier. a schematic representation of the three strategies with the corresponding results is shown in figure after normalisation of the data so as to correct for differences in transfection efficiencies the results show that all three strategies result in a comparable trans-activation of gal4-pzsgreen as a result of ar-lbd and ar-ntd interaction. although prb slides offer a cost-effective and robust platform for simultaneous comparison of large number of interactions in different cell lines and under different culture conditions, the pr slides are more suitable for the large-scale screening of novel bait-prey interactions. as shown in additional file the spot to spot distance on cappia slides can easily be reduced to mm. by printing each sample as one feature per slide and using a spot distance of mm, up to different preys can be printed on one slide. replicate pr-slides can then be screened with any bait of interest, further increasing the high-throughput application of cappia. in addition pr slides can be printed in large batches and can be stored for at least months at 4°c and even longer at -80°c, thus increasing the cost effectiveness of cappia. the slides can be shipped on ice, allowing any research group with access to standard culture facilities to interrogate the prey library with their bait of interest. discussion we hereby present a new screening technology for the high-throughput interrogation of protein-protein interactions in mammalian cells. this cell-array-protein-protein-interaction-assay combines the advantages of mammalian two-hybrid systems with those of microarrays, a combination that is expected to save considerable time and expense. indeed, cappia slides are printed with the same robotic microarray devices used to print conventional dna microarrays. consequently, cell arrays require far less dna, transfection reagents and cells as compared to assays performed in microwell plate format. for comparison, the amount of transfection reagent required to transfect one sample in a micro-well is sufficient to transfect more than samples in cappia, a significant cost reduction since transfection reagents account for a major part of the expenses in these assays. although cell arrays can theoretically carry up to features per slide, the actual number of features per slide is dependent on the type of application. since the efficiency of the simultaneous transfection of three plasmids is lower than that of a single plasmid and since the reporter will only be expressed after sufficient amounts of both prey and bait proteins are expressed in the same cell there is a need to increase the number of cells per spot in cappia in order to guarantee the robust detection of interacting proteins. consequently, in order to increase the sensitivity of the assay, the features on cappia slides need to be bigger and hence the slide capacity will be lower. still, cappia slides can be printed with each containing up to different preys as single spots when using a spot to spot distance of mm. this is equivalent to standard micro-well plates and data can then be collected from a number of identical slides to obtain replicate data points per sample. in addition, by pooling preys per feature even more preys can be interrogated per slide. the cost-effectiveness of the assay is further increased by using an auto-fluorescent based reporter eliminating the need for immuno-fluorescence staining or enzyme-based reporter detection. in contrast to microwell-based assays no extensive liquid handling infrastructure is required once the slides have been printed. in addition pr or prb slides can be printed in large batches and stored frozen without losing their efficiency, thus increasing the flexibility and cost effectiveness of cappia. finally, pr slides can be screened with any bait of interest simply by using standard transfection and cell culture methods, available to many research groups. compared to the more common high-throughput yeast two-hybrid assay, cappia offers the advantage of testing mammalian ppi within a cellular context that more closely mimics the native protein environment. as a consequence the rate of false negative interactions, i.e. true interactions that can not be detected due to the inappropriate folding and/or post-translational processing of the proteins should be lower using cappia, especially since phenotypically different cell types can be used. as with dna microarrays, pr-slides interrogated with different baits allow a comparison between multiple identical assays. as such preys or baits that are found multiple times with unrelated proteins, so-called bait- or prey-unspecific false positives can be distinguished without need for further experimentation. high-throughput yeast two-hybrid assays also require extensive liquid handling infrastructures making the assays less accessible for many research groups. previously, suzuki et al described a pcr-mediated rapid sample preparation and a high-throughput assay system based on the mammalian two-hybrid method. although the method they designed allows for the rapid preparation of high numbers of bait and prey samples, the actual two-hybrid assays are performed in microwells and require semiautomatic multiple dispensers as well as multiple reagents for downstream enzymatic detection of interacting proteins. cappia, on the other hand allows the quantitative analysis of interactions comparable to assays performed in microwells, while offering the advantage of being economical and accessible to research groups equipped with basic cell culture infrastructures. currently, large bait-prey libraries for use in cappia are being generated using gateway compatible destination pcmv-ad and pcmv-bd vectors and panels of pentry clones. since cappia is based on the gal4-two-hybrid system that is already used by many research groups, existing baits and preys can easily be included in the assays. in particular, the trans-bait version of cappia readily allows available bait constructs to be screened against large number of preys.  taken together, the high capacity of the cell arrays, the flexibility to interrogate any bait of interest and the small amounts of reagents that are required make cappia currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells. microarray-based systems such as the one presented here should lead to the acceleration of the deciphering of functional protein complexes. in addition cappia could also be an economical alternative for the identification of potential drug targets by combining screens for small molecule ligands that can disrupt or modulate interactions with selected prey-bait combinations expressed in array format inside micro-wells. 
using known interaction partners and by screening different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. the high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells. investigation of protein-protein interactions is therefore essential for understanding of cellular functions. we here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of dna microarrays. in this cell array protein-protein interaction assay, mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. most of the biological processes rely on the formation of protein complexes. we demonstrate that the cappia assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. it is advantageous to perform mammalian ppi analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. until now mammalian two-hybrid assays have been performed on individual gene scale.
 most of the biological processes rely on the formation of protein complexes. investigation of protein-protein interactions is therefore essential for understanding of cellular functions. it is advantageous to perform mammalian ppi analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. until now mammalian two-hybrid assays have been performed on individual gene scale. we here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of dna microarrays.  in this cell array protein-protein interaction assay, mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter. using known interaction partners and by screening different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. moreover, different strategies in respect to bait-prey combinations are presented.  we demonstrate that the cappia assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. the high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells. 