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Protein Microarray Technology

David A. Hall, Jason Ptacek , and Michael Snyder   

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Abstract

Protein chips have emerged as a promising approach for a wide variety of applications including

the identification of protein-protein interactions, protein-phospholipid interactions, smallmolecule targets, and substrates of proteins kinases. They can also be used for clinical

diagnostics and monitoring disease states. This article reviews current methods in the generation

and applications of protein microarrays.

Understanding complex cellular systems will require the identification and analysis of each of its

components and determining how they function together and are regulated. A critical step in this process is to determine the biochemical activities of the proteins and how these activities

themselves are controlled and modified by other proteins. Traditionally, the biochemical

activities of proteins have been elucidated by studying single molecules, one experiment at a

time. This process is not optimal, as it is slow and labor intensive.

In contrast to this traditional approach, high-throughput scientific methods have been developed

in the last decade to optimize the study of large numbers of molecules, including DNA, proteinsand metabolites. DNA microarrays in particular have proved valuable in genomic research

(Schena et al., 1995). They have been used to study gene expression patterns, to locatetranscription factor binding sites, and to detect sequence mutations and deletions on a grandscale. However, DNA microarrays tell us only about the genes themselves and provide little

information regarding the functions of the proteins they encode. More recently high throughput

approaches have been developed for the study of proteins, including profiling of proteins usingmass spectrometry (Gavin et al., 2006; Gavin et al., 2002; Ho et al., 2002; Krogan et al., 2006; 

Washburn et al., 2001), tagging and subcellular localization (Ghaemmaghami et al., 2003; Huh et

al., 2003) and protein microarrays (MacBeath and Schreiber, 2000; Zhu et al., 2001; Zhu et al.,2000). This article reviews the developments in the production and application of protein

microarrays.

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Types of protein microarrays

Three types of protein microarrays are currently used to study the biochemical activities of

 proteins: analytical microarrays, functional microarrays, and reverse phase microarrays.

Analytical microarrays are typically used to profile a complex mixture of proteins in order to

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measure binding affinities, specificities, and protein expression levels of the proteins in the

mixture. In this technique, a library of antibodies, aptamers, or affibodies is arrayed on a glass

microscope slide. The array is then probed with a protein solution. Antibody microarrays are themost common analytical microarray (Bertone and Snyder, 2005).

These types of microarrays can be used to monitor differential expression profiles and forclinical diagnostics. Examples include profiling responses to environmental stress and healthy

versus disease tissues (Sreekumar et al., 2001).

Functional protein microarrays differ from analytical arrays in that functional protein arrays are

composed of arrays containing full-length functional proteins or protein domains. These protein

chips are used to study the biochemical activities of an entire proteome in a single experiment.They are used to study numerous protein interactions, such as protein-protein, protein-DNA,

 protein-RNA, protein-phospholipid, and protein-small molecule interactions (Hall et al., 2004; 

Zhu et al., 2001).

A third type of protein microarray, related to analytical microarrays, is known as a reverse phase protein microarray (RPA). In RPA, cells are isolated from various tissues of interest and are

lysed. The lysate is arrayed onto a nitrocellulose slide using a contact pin microarrayer. Theslides are then probed with antibodies against the target protein of interest, and the antibodies are

typically detected with chemiluminescent, fluorescent, or colorimetric assays. Reference peptides

are printed on the slides to allow for protein quantification of the sample lysates.

RPAs allow for the determination of the presence of altered proteins that may be the result of

disease. Specifically, post-translational modifications, which are typically altered as a result ofdisease, can be detected using RPAs (Speer et al., 2005). Once it is determined which protein

 pathway may be dysfunctional in the cell, a specific therapy can be determined to target the

dysfunctional protein pathway and treat the disease of interest.

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Proteome libraries

Challenges to creating a proteome microarray include not only the creation of the necessaryover-expression library, but also the development of a high-throughput expression and

 purification protocol necessary to efficiently produce thousands of pure, functional proteins that

can be immobilized onto a solid surface. There are two major recombinational cloning strategies

that are used to create libraries of open reading frames (ORFs) in expression vectors (Phizicky et

al., 2003). The first makes use of recombination in yeast. Open reading frames of interest areamplif ied such that they all contain common 5′ and different common 3′ ends. The ORFs are

mixed with a linearized vector that contains ends identical to the 5′ and 3′ ends of the amplified

ORFs. The mixture is then transformed into yeast where gap-mediated recombination occurs(Phizicky et al., 2003). The first yeast proteome collection was produced in this fashion (Zhu et

al., 2001).

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The other major cloning strategy makes use of the Gateway recombinational cloning system

(Phizicky et al., 2003). The Gateway system takes advantage of the integration and excision

 properties of phage λ in E. coli. λ phage integrates into its hosts DNA by recombination betweenthe λ att  P site and the host att  B site. The recombination results in DNA with ends called att  L

and att  R. Upon excision of the λ phage, recombination between att  L and att  R results in the

original att  P and att  B sites. The Gateway system takes advantage of these unique properties ofλ phage to create an expression vector with the ORF of interest via a 2 step recombinationreaction. First, components of λ integration are mixed with the amplified ORF that contains 2

different directional att  B sites. This mixture is then combined, in vitro, with a vector that

contains corresponding att  P sites. Recombination occurs and the resulting vector then containstwo different att  L sites. This vector is then combined with an expression vector that contains

appropriate att  R sites. Recombination occurs in vitro, and the ORF of interest is then in a useful

expression vector. The Gateway system is quite flexible as it allows for easy shuttling of ORFs

 between different expression vectors. The Gateway system has been used to create ORFcollections for genes from yeast (Gelperin et al., 2005), C. elegans (Reboul et al., 2003), and

humans (Rual et al., 2004b).

The generation of the first proteome library and its use in microarrays were performed for yeast.

The first library contains greater than 5800 yeast proteins, which are tagged at their N-terminuswith GST-HisX6 for ease of purification. After expressing and purifying the proteins in a high-throughput fashion in budding yeast, the proteins were spotted onto glass slides (Zhu et al.,

2001). Protein signals on the chips were quantified by probing the GST-HisX6 tags with anti-

GST antibodies, followed by incubation with fluorophore-conjugated secondary antibodies.Protein-protein and protein-lipid experiments conducted on the chip led to the discovery of new

calmodulin and phospholipid binding proteins and were proof that the immobilized proteins on

the array were indeed active.

More recently, Gelperin and colleagues have created a yeast proteome library of C-terminally

TAP-tagged proteins which allows for the more efficient purification of transmembrane and

secreted proteins (Gelperin et al., 2005). Gelperin and colleagues’ collection makes use of theGateway recombinational technology, which allows for the easy transfer of ORFs into a variety

of expression vectors.

In addition to existing proteome collections in yeast, efforts such as the ORFeome project are

underway to create clone collections in higher eukaryotes (Brasch et al., 2004; Reboul et al.,

2003; Rual et al., 2004a; Rual et al., 2004b). Vidal and colleagues define the ORFeome as the setof protein encoding open reading frames for an entire organism. They have used Gateway

recombinational cloning technology (Hartley et al., 2000) to create the first C. elegans ORFeome

(Reboul et al., 2003), and a first version of the human ORFeome (Rual et al., 2004b). Human

ORFeome collections and microarrays have also been generated by Invitrogen. These ORFeomesare helpful in improving the annotation of genomes, and because they were created using

Gateway technology, they exist in a flexible format that allows for the high-throughput

expression of proteins in a number of different experimental systems.

Successful ORF cloning in higher eukaryotes is dependent on full length cDNA collections, and

there are numerous public efforts underway to create human full length cDNA collections, such

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as the Unigene set (Wheeler et al., 2004), the Full Length Expression (FLEX) Gene repository

(Brizuela et al., 2001), the Integrated Molecular Analysis of Genomes and their Expression

(IMAGE) cDNA collection (Lennon et al., 1996), and the Mammalian Gene Collection (MGC)(Gerhard et al., 2004). Commercial collections are also available from companies such as

Invitrogen, GeneCopoeia, OriGene, and Open Biosystems.

A protein microarray is only as useful as the quality of the proteins arrayed on the chip. The

 production of proteins in homologous systems (i.e. yeast protein expressed in yeast) is expected

to greatly improve the quality of the proteins and produce them in an active state. Because thefunction of many if not most proteins are not known, it is impossible to completely determine

whether every protein on the array is active and functional. It is likely, however, that for a large

majority of the proteins on the yeast proteome chips at least some material is functional, because

a number of successful biochemical activities have been confirmed and discovered using our proteome chips. These biochemical activities include protein-protein, protein-phospholipid,

 protein-DNA, and protein-small molecule interactions (Hall et al., 2004; Huang et al., 2004; Zhu

et al., 2001), as well as enzymatic reactions (Ptacek et al., 2005; Zhu et al., 2000) (Figure 1).

Figure 1 

Applications of functional protein microarrays 

It is possible that the position of the affinity tags used for purification may interfere with thefunctionality of the proteins. The use of two yeast proteome collections, one with C-terminal

tags, and one with a different N-terminal tag is expected to complement each other (Gelperin etal., 2005; Zhu et al., 2001). If one version of the tagged protein exhibits a loss of functionality

due to the location of the tag, then the other tagged version of the protein can be used on the

chip.

Recently a method has been described for the direct production of proteins on chips. DNA is

spotted on a microscope slide and subjected to an in vitro transcription and translation system.The proteins are produced as GST fusions and adhere to glutathione on the surface of the slide.

Using this system LaBaer and coworkers have produced a number of human proteins involved in

DNA metabolism and demonstrated protein-protein interactions (Ramachandran et al., 2004).

This method is advantageous because it produces protein directly on the slide without requiring

 purification and that proteins do not need to be stored.

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Protein chips

Typically, protein chips are prepared by immobilizing proteins onto a treated microscope slide

using a contact spotter (MacBeath and Schreiber, 2000; Zhu et al., 2001) or a non-contact

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microarrayer (Delehanty, 2004; Delehanty and Ligler, 2003; Jones et al., 2006). It is critical that

the proteins remain in a wet environment. Thus, sample buffers contain a high percent of

glycerol, and the printing process is carried out in a humidity-controlled environment (MacBeathand Schreiber, 2000; Zhu et al., 2001). Because equipment and procedures developed for DNA

microarrays are easily adaptable to the development of protein microarrays, the choice of using

treated microscope slides follows from the ready availability of robotic arrayers and laserscanners that have become commonplace in the world of DNA microarrays (Bertone and Snyder,2005).

A number of different slide surfaces can be used for protein chips. In choosing a slide surface,

the goals should be immobilizing the protein on the chip, maintaining the conformation and the

functionality of the protein, and achieving maximum binding capacity (Zhu et al., 2003). It is

also important to consider whether a random or a uniform orientation of proteins on the slidesurface is desired (Figure 2). For random attachment of proteins through amines, aldehyde- and

epoxy-derivatized glass surfaces can be used (Kusnezow et al., 2003; MacBeath and Schreiber,

2000). Coating the glass surface with nitrocellulose, gel pads, or poly-L-lysine also achieves a

random orientation of the proteins as the proteins are passively adsorbed onto the surface(Angenendt et al., 2002; Charles et al., 2004; Kramer et al., 2004; Stillman and Tonkinson, 2000; 

Zhu et al., 2003).

Figure 2 

Protein attachment methods 

Affinity tag surfaces can be used for the uniform orientation of proteins on the chip surface. One popular slide choice is the nickel coated slide for use with HisX6 tagged proteins (Zhu et al.,

2003). Another is streptavidin coated slides (Lesaicherre et al., 2002). The display of the proteinsaway from the chip surface should give reagents easier access to the active sites of the proteins

(Zhu et al., 2003).

Finally microwells can also be used for protein assays. Zhu and colleagues developed a slide

surface which contains microwells made of a silicone elastomer and attached protein to the

epoxy-treated microwells (Zhu et al., 2000). The benefit of microwells is the ability to performexperiments in an aqueous environment while preventing cross contamination.

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Detection methods

To locate reactive proteins on a proteome chip, small molecule probes are labeled with either

fluorescent, affinity, photochemical, or radioisotope tags. Fluorescent labels are generally

 preferred, as they are safe and effective and are compatible with readily available microarray

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laser scanners. However, probes can also be labeled with affinity tags or photochemical tags

(Colca and Harrigan, 2004; Mitsopoulos et al., 2004). Huang and colleagues used biotin labeled

small molecules to probe proteome chips for small molecule inhibitors of rapamycin (Huang etal., 2004). The reactive proteins were identified with Cy3 labeled streptavidin.

Regardless of the type of label used, there are problems associated with labeling the moleculesused to probe a proteome chip. Chief among these problems is the possibility that the label itself

may interfere with the probe’s ability to interact with the target protein. To overcome this

 problem, a number of label-free detection methods have recently been developed. Label-freedetection methods not only overcome the problem of steric hindrance of a label, but also allow

for the collection of kinetic binding data (reviewed in (Ramachandran et al., 2005)).

The current leading technology for label-free detection of protein interactions is surface plasmon

resonance (SPR), which probes the local index of refraction. Other choices include carbon

nanotubes, carbon nanowires, and microelectromechanical systems cantilevers. While these

technologies are still in their infancy and are not suitable for high-throughput protein interaction

detections, they do offer much promise (Ramachandran et al., 2005).

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Applications of protein chips

The biochemistries of thousands of proteins can be characterized and quantified in a parallel

format through the use of protein microarrays. Not only have protein chips been used to

characterize the functions of previously uncharacterized proteins, they have also been used to

discover new functionalities for previously characterized proteins. Proteome chips have beenused to study protein-protein interactions (Zhu et al., 2001), protein-DNA interactions (Hall et

al., 2004), protein-lipid interactions (Zhu et al., 2001), protein-drug interactions (Huang et al.,2004), protein-receptor interactions (Jones et al., 2006), and antigen-antibody interactions(Michaud et al., 2003). In addition, proteome chips have been used to study kinase activities

(Ptacek et al., 2005; Zhu et al., 2000) and have been used for serum profiling (Zhu et al., 2006).

With respect to protein-protein interactions, yeast proteome chips have been used to study

calmodulin binding proteins (Zhu et al., 2001). Calmodulin is a calcium binding protein involved

in many calcium-regulated cellular pathways (Hook and Means, 2001). Zhu and colleagues biotinylated the calmodulin probes, and detected the protein-protein interactions using Cy3-

labeled streptavidin. Their study found 6 known calmodulin binding proteins, and 33 additional

 potential binders. Their study also revealed a novel consensus binding motif that was related to a

 previously known calmodulin binding motif (Zhu et al., 2001).

Hall et al. used yeast proteome chips to identify previously unrecognized DNA binding activities

(Hall et al., 2004) (Figure 3). Both single and double stranded Cy3 labeled yeast genomic DNAwas used to probe a yeast proteome array. Over 200 DNA binding proteins were identified, but

only about half of those were expected to bind DNA based on their known function. One of the

unexpected targets we found was Arg5,6, a mitochondrial enzyme involved in arginine biosynthesis. Chromatin immunoprecipitation experiments revealed that Arg5,6 associates with

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specific nuclear and mitochondrial loci in vivo. Gel shift assays revealed that Arg5,6 binds to

specific DNA fragments in vitro, and a common binding motif was found. Real time PCR

experiments with Arg5,6 indicated that it may have a role in regulating gene expression. Thus, ayeast proteome chip was used to discover a novel DNA binding protein, and a metabolic enzyme

was found that appears to have a role in directly regulating eukaryotic gene expression.

Figure 3 

Identification of DNA-binding proteins using a functional protein microarray 

Zhu and colleagues also used yeast proteome chips to study phosphoinositide (PI) binding proteins (Zhu et al., 2001). Phosphoinositides are constituents of cellular membranes and also

regulate a number of diverse cellular processes (Fruman et al., 1998; Odorizzi et al., 2000).Biotinylated PI liposomes were used to probe the chip. They were detected using Cy-3 labeled

streptavidin. Of the 150 novel lipid binding proteins that were identified, 52 corresponded touncharacterized proteins, and 45 were membrane associated proteins.

Proteome microarrays are also an excellent way to discover drug targets. Entire proteomes printed on a chip can be probed with small molecules in one experiment to discover interactions.

Huang and colleagues used yeast proteome microarrays to study protein-drug interactions

(Huang et al., 2004). They probed the array with a biotinylated small molecule inhibitor of

rapamycin (SMIR) to find protein targets that may be involved in the target-of-rapamycin (TOR)dependent nutrient response network. They discovered a protein of previously unknown function

to be a target of the SMIR. They followed up the array probing experiment with gene deletion

experiments to prove that the target protein was indeed a legitimate target of the SMIR.

Jones and colleagues have used protein microarrays to study protein recruitment to receptors in a

high-throughput fashion. Using the data obtained from the microarrays, they also calculated thedissociation constants of the protein-receptor binding (Jones et al., 2006). Specifically, they

created protein microarrays containing most of the human Src homology 2 (SH2) and

 phosphotyrosine binding (PTB) domains. These binding domains are known to interact with

different epidermal growth factor receptors (EGFR), which themselves are involved in a widevariety of cellular responses. The activated receptors become phosphorylated on their tyrosine

residues, which then become the sites for the SH2 and PTB domain binding. The human binding

domain microarray of 159 proteins was probed with 66 fluorescently labeled peptides

representing the binding sites of the epidermal growth factor receptors. The microarrays were

 probed with eight concentrations of each peptide, and the resulting fluorescence data was used tocalculate dissociation constants to determine the binding affinities of the peptide-binding domain

interactions. The accuracy of a sample of the dissociation constants calculations was confirmedwith surface plasmon resonance experiments. From their experimental data, they constructed a

quantitative EGFR interaction map. Their data not only confirmed previously known

interactions, but also showed novel biophysical interactions of SHT2 and PTB domains with theepidermal growth factor receptor network. With their quantitative data, they found that different

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receptor tyrosine kinases differ in their level of selectivity when overexpressed, which they

suggest may be a clue as to why some receptors have more oncogenic potential than others.

There have been multiple yeast protein kinase studies using protein chips. Zhu and colleagues

used silicone elastomer nanowell sheets placed onto glass slides to study the activity of 119 yeast

kinases using 17 different substrates (Zhu et al., 2000). The 119 kinases were overexpressed andcovalently attached to the nanowell chip. The kinases were incubated with 17 different substrates

along with radio labeled ATP to test for in vitro kinase activity. They obtained many novel

results, including that 27 yeast kinases can act in vitro as tyrosine kinases. This is roughly triplethe number of tyrosine kinases originally thought to exist in yeast.

Ptacek and colleagues have also studied protein phosphorylation in yeast using proteome chips(Ptacek et al., 2005). Their goal was to develop a global kinase-substrate map for yeast. To this

end, they incubated 87 different yeast protein kinases or kinase complexes separately with radio

labeled ATP on a yeast proteome chip containing 4400 unique proteins (Figure 4). They found

about 4200 phosphorylation events affecting 1325 different proteins, and from this data

assembled an in vitro phosphorylation network.

Figure 4 A. A yeast proteome microarray containing ~4,400 GST-tagged yeast proteins printed in

duplicate. The slide was probed with anti-GST antibodies followed by Cy5-labeled anti-rabbit

antibodies. 40 of 48 blocks are shown.

Proteome chips have also been used successfully to screen patient’s sera for the presence ofautoantibodies (Hueber et al., 2005; Kattah et al., 2006) or viral specific antibodies (Zhu et al.,

2006). Zhu and colleagues created a coronavirus protein microarray and used the array to rapidlyscreen patient’s sera for the presence of antibodies against the SARS-CoV coronavirus (Zhu et

al., 2006). They fabricated a protein microarray using coronavirus proteins overexpressed in

yeast. The proteins were spotted in duplicate onto microscope slides. Human serum samplesfrom SARS infected or healthy subjects were screened using the protein chips, and bound

antibodies were detected fluorescently using Cy3-labelled anti-human IgG or IgM antibodies.

They found that their coronavirus protein arrays were able to accurately diagnose the presence ofantibodies against coronaviruses in greater than 90% of the patient serum samples they tested.

Accordingly, the proteome array they created was an excellent way to quickly diagnose those

 patients displaying symptoms of SARS infection.

Another set of antigen-antibody experiments using proteome microarrays was performed by

Michaud and colleagues (Michaud et al., 2003). To analyze antibody specificity, they screened

11 monoclonal and polyclonal antibodies against a yeast proteome microarray made up ofapproximately 5000 different yeast proteins. Fluorescent detection was performed using

secondary antibodies labeled with Cy5. They found varying degrees of cross-reactivity among

the antibodies that could not necessarily be predicted according to the amino acid sequences of

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the proteins in question. Their data should prove useful in evaluating assays using antibodies

included in their study.

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Conclusion

Proteome chip technology is an excellent high-throughput method used to probe an entire

collection of proteins for a specific function or biochemistry. It is an exceptional new way todiscover previously unknown multifunctional proteins, and to discover new functionalities for

well-studied proteins.

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Acknowledgments

We thank Heng Zhu for comments on the manuscript.

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Footnotes

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50. 51. Did you mean: Protein Microarray Technology David A. Hall, Jason Ptacek, and Michael

Snyder Corresponding  author Author information ► Copyright and License information

► The publisher's final edited version of this article is available at Mech Ageing Dev Seeother articles in PMC that cite the published article. Go to: Abstract Protein chips have

emerged as a promising approach for a wide variety of applications including the

identification of protein-protein interactions, protein-phospholipid interactions, smallmolecule targets, and substrates of proteins kinases. They can also be used for clinicaldiagnostics and monitoring disease states. This article reviews current methods in the

generation and applications of protein microarrays. Understanding complex cellular

systems will require the identification and analysis of each of its components and

determining how they function together and are regulated. A critical step in this processis to determine the biochemical activities of the proteins and how these activities

themselves are controlled and modified by other proteins. Traditionally, the biochemical

activities of proteins have been elucidated by studying single molecules, one experimentat a time. This process is not optimal, as it is slow and labor intensive. In contrast to this

traditional approach, high-throughput scientific methods have been developed in the last

decade to optimize the study of large numbers of molecules, including DNA, proteins andmetabolites. DNA microarrays in particular have proved valuable in genomic research

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(Schena et al., 1995). They have been used to study gene expression patterns, to locate

transcription factor binding sites, and to detect sequence mutations and deletions on a

grand scale. However, DNA microarrays tell us only about the genes themselves and provide little information regarding the functions of the proteins they encode. More

recently high throughput approaches have been developed for the study of proteins,

including profiling of proteins using mass spectrometry (Gavin et al., 2006; Gavin et al.,2002; Ho et al., 2002; Krogan et al., 2006; Washburn et al., 2001), tagging andsubcellular localization (Ghaemmaghami et al., 2003; Huh et al., 2003) and protein

microarrays (MacBeath and Schreiber, 2000; Zhu et al., 2001; Zhu et al., 2000). This

article reviews the developments in the production and application of proteinmicroarrays. Go to: Types of protein microarrays Three types of protein microarrays are

currently used to study the biochemical activities of proteins: analytical microarrays,

functional microarrays, and reverse phase microarrays. Analytical microarrays are

typically used to profile a complex mixture of proteins in order to measure bindingaffinities, specificities, and protein expression levels of the proteins in the mixture. In this

technique, a library of antibodies, aptamers, or affibodies is arrayed on a glass

microscope slide. The array is then probed with a protein solution. Antibody microarraysare the most common analytical microarray (Bertone and Snyder, 2005). These types of

microarrays can be used to monitor differential expression profiles and for clinical

diagnostics. Examples include profiling responses to environmental stress and healthy

versus disease tissues (Sreekumar et al., 2001). Functional protein microarrays differfrom analytical arrays in that functional protein arrays are composed of arrays containing

full-length functional proteins or protein domains. These protein chips are used to study

the biochemical activities of an entire proteome in a single experiment. They are used tostudy numerous protein interactions, such as protein-protein, protein-DNA, protein-RNA,

 protein-phospholipid, and protein-small molecule interactions (Hall et al., 2004; Zhu et

al., 2001). A third type of protein microarray, related to analytical microarrays, is known

as a reverse phase protein microarray (RPA). In RPA, cells are isolated from varioustissues of interest and are lysed. The lysate is arrayed onto a nitrocellulose slide using a

contact pin microarrayer. The slides are then probed with antibodies against the target

 protein of interest, and the antibodies are typically detected with chemiluminescent,fluorescent, or colorimetric assays. Reference peptides are printed on the slides to allow

for protein quantification of the sample lysates. RPAs allow for the determination of the

 presence of altered proteins that may be the result of disease. Specifically, post-translational modifications, which are typically altered as a result of disease, can be

detected using RPAs (Speer et al., 2005). Once it is determined which protein pathway

may be dysfunctional in the cell, a specific therapy can be determined to target the

dysfunctional protein pathway and treat the disease of interest. Go to: Proteome librariesChallenges to creating a proteome microarray include not only the creation of the

necessary over-expression library, but also the development of a high-throughput

expression and purification protocol necessary to efficiently produce thousands of pure,

functional proteins that can be immobilized onto a solid surface. There are two majorrecombinational cloning strategies that are used to create libraries of open reading frames

(ORFs) in expression vectors (Phizicky et al., 2003). The first makes use of

recombination in yeast. Open reading frames of interest are amplified such that they allcontain common 5′ and different common 3′ ends. The ORFs are mixed with a linearized

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vector that contains ends identical to the 5′ and 3′ ends of the amplified ORFs. The

mixture is then transformed into yeast where gap-mediated recombination occurs

(Phizicky et al., 2003). The first yeast proteome collection was produced in this fashion(Zhu et al., 2001). The other major cloning strategy makes use of the Gateway

recombinational cloning system (Phizicky et al., 2003). The Gateway system takes

advantage of the integration and excision properties of phage λ in E. coli. λ phageintegrates into its hosts DNA by recombination between the λ att P site and the host att Bsite. The recombination results in DNA with ends called att L and att R. Upon excision of

the λ phage, recombination between att L and att R results in the original att P and att B

sites. The Gateway system takes advantage of these unique properties of λ phage to createan expression vector with the ORF of interest via a 2 step recombination reaction. First,

components of λ integration are mixed with the amplified ORF that contains 2 different

directional att B sites. This mixture is then combined, in vitro, with a vector that contains

corresponding att P sites. Recombination occurs and the resulting vector then containstwo different att L sites. This vector is then combined with an expression vector that

contains appropriate att R sites. Recombination occurs in vitro, and the ORF of interest is

then in a useful expression vector. The Gateway system is quite flexible as it allows foreasy shuttling of ORFs between different expression vectors. The Gateway system has

 been used to create ORF collections for genes from yeast (Gelperin et al., 2005), C.

elegans (Reboul et al., 2003), and humans (Rual et al., 2004b). The generation of the first

 proteome library and its use in microarrays were performed for yeast. The first librarycontains greater than 5800 yeast proteins, which are tagged at their N-terminus with

GST-HisX6 for ease of purification. After expressing and purifying the proteins in a

high-throughput fashion in budding yeast, the proteins were spotted onto glass slides(Zhu et al., 2001). Protein signals on the chips were quantified by probing the GST-

HisX6 tags with anti-GST antibodies, followed by incubation with fluorophore-

conjugated secondary antibodies. Protein-protein and protein-lipid experiments

conducted on the chip led to the discovery of new calmodulin and phospholipid binding proteins and were proof that the immobilized proteins on the array were indeed active.

More recently, Gelperin and colleagues have created a yeast proteome library of C-

terminally TAP-tagged proteins which allows for the more efficient purification oftransmembrane and secreted proteins (Gelperin et al., 2005). Gelperin and colleagues’

collection makes use of the Gateway recombinational technology, which allows for the

easy transfer of ORFs into a variety of expression vectors. In addition to existing proteome collections in yeast, efforts such as the ORFeome project are underway to

create clone collections in higher eukaryotes (Brasch et al., 2004; Reboul et al., 2003;

Rual et al., 2004a; Rual et al., 2004b). Vidal and colleagues define the ORFeome as the

set of protein encoding open reading frames for an entire organism. They have usedGateway recombinational cloning technology (Hartley et al., 2000) to create the first C.

elegans ORFeome (Reboul et al., 2003), and a first version of the human ORFeome (Rual

et al., 2004b). Human ORFeome collections and microarrays have also been generated by

Invitrogen. These ORFeomes are helpful in improving the annotation of genomes, and because they were created using Gateway technology, they exist in a flexible format that

allows for the high-throughput expression of proteins in a number of different

experimental systems. Successful ORF cloning in higher eukaryotes is dependent on fulllength cDNA collections, and there are numerous public efforts underway to create

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human full length cDNA collections, such as the Unigene set (Wheeler et al., 2004), the

Full Length Expression (FLEX) Gene repository (Brizuela et al., 2001), the Integrated

Molecular Analysis of Genomes and their Expression (IMAGE) cDNA collection(Lennon et al., 1996), and the Mammalian Gene Collection (MGC) (Gerhard et al.,

2004). Commercial collections are also available from companies such as Invitrogen,

GeneCopoeia, OriGene, and Open Biosystems. A protein microarray is only as useful asthe quality of the proteins arrayed on the chip. The production of proteins in homologoussystems (i.e. yeast protein expressed in yeast) is expected to greatly improve the quality

of the proteins and produce them in an active state. Because the function of many if not

most proteins are not known, it is impossible to completely determine whether every protein on the array is active and functional. It is likely, however, that for a large majority

of the proteins on the yeast proteome chips at least some material is functional, because a

number of successful biochemical activities have been confirmed and discovered using

our proteome chips. These biochemical activities include protein-protein, protein- phospholipid, protein-DNA, and protein-small molecule interactions (Hall et al., 2004;

Huang et al., 2004; Zhu et al., 2001), as well as enzymatic reactions (Ptacek et al., 2005;

Zhu et al., 2000) (Figure 1). Figure 1 Figure 1 Applications of functional proteinmicroarrays It is possible that the position of the affinity tags used for purification may

interfere with the functionality of the proteins. The use of two yeast proteome collections,

one with C-terminal tags, and one with a different N-terminal tag is expected to

complement each other (Gelperin et al., 2005; Zhu et al., 2001). If one version of thetagged protein exhibits a loss of functionality due to the location of the tag, then the other

tagged version of the protein can be used on the chip. Recently a method has been

described for the direct production of proteins on chips. DNA is spotted on a microscopeslide and subjected to an in vitro transcription and translation system. The proteins are

 produced as GST fusions and adhere to glutathione on the surface of the slide. Using this

system LaBaer and coworkers have produced a number of human proteins involved in

DNA metabolism and demonstrated protein-protein interactions (Ramachandran et al.,2004). This method is advantageous because it produces protein directly on the slide

without requiring purification and that proteins do not need to be stored. Go to: Protein

chips Typically, protein chips are prepared by immobilizing proteins onto a treatedmicroscope slide using a contact spotter (MacBeath and Schreiber, 2000; Zhu et al.,

2001) or a non-contact microarrayer (Delehanty, 2004; Delehanty and Ligler, 2003; Jones

et al., 2006). It is critical that the proteins remain in a wet environment. Thus, sample buffers contain a high percent of glycerol, and the printing process is carried out in a

humidity-controlled environment (MacBeath and Schreiber, 2000; Zhu et al., 2001).

Because equipment and procedures developed for DNA microarrays are easily adaptable

to the development of protein microarrays, the choice of using treated microscope slidesfollows from the ready availability of robotic arrayers and laser scanners that have

 become commonplace in the world of DNA microarrays (Bertone and Snyder, 2005). A

number of different slide surfaces can be used for protein chips. In choosing a slide

surface, the goals should be immobilizing the protein on the chip, maintaining theconformation and the functionality of the protein, and achieving maximum binding

capacity (Zhu et al., 2003). It is also important to consider whether a random or a uniform

orientation of proteins on the slide surface is desired (Figure 2). For random attachmentof proteins through amines, aldehyde- and epoxy-derivatized glass surfaces can be used

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(Kusnezow et al., 2003; MacBeath and Schreiber, 2000). Coating the glass surface with

nitrocellulose, gel pads, or poly-L-lysine also achieves a random orientation of the

 proteins as the proteins are passively adsorbed onto the surface (Angenendt et al., 2002;Charles et al., 2004; Kramer et al., 2004; Stillman and Tonkinson, 2000; Zhu et al.,

2003). Figure 2 Figure 2 Protein attachment methods Affinity tag surfaces can be used

for the uniform orientation of proteins on the chip surface. One popular slide choice is thenickel coated slide for use with HisX6 tagged proteins (Zhu et al., 2003). Another isstreptavidin coated slides (Lesaicherre et al., 2002). The display of the proteins away

from the chip surface should give reagents easier access to the active sites of the proteins

(Zhu et al., 2003). Finally microwells can also be used for protein assays. Zhu andcolleagues developed a slide surface which contains microwells made of a silicone

elastomer and attached protein to the epoxy-treated microwells (Zhu et al., 2000). The

 benefit of microwells is the ability to perform experiments in an aqueous environment

while preventing cross contamination. Go to: Detection methods To locate reactive proteins on a proteome chip, small molecule probes are labeled with either fluorescent,

affinity, photochemical, or radioisotope tags. Fluorescent labels are generally preferred,

as they are safe and effective and are compatible with readily available microarray laserscanners. However, probes can also be labeled with affinity tags or photochemical tags

(Colca and Harrigan, 2004; Mitsopoulos et al., 2004). Huang and colleagues used biotin

labeled small molecules to probe proteome chips for small molecule inhibitors of

rapamycin (Huang et al., 2004). The reactive proteins were identified with Cy3 labeledstreptavidin. Regardless of the type of label used, there are problems associated with

labeling the molecules used to probe a proteome chip. Chief among these problems is the

 possibility that the label itself may interfere with the probe’s ability to interact with thetarget protein. To overcome this problem, a number of label-free detection methods have

recently been developed. Label-free detection methods not only overcome the problem of

steric hindrance of a label, but also allow for the collection of kinetic binding data

(reviewed in (Ramachandran et al., 2005)). The current leading technology for label-freedetection of protein interactions is surface plasmon resonance (SPR), which probes the

local index of refraction. Other choices include carbon nanotubes, carbon nanowires, and

microelectromechanical systems cantilevers. While these technologies are still in theirinfancy and are not suitable for high-throughput protein interaction detections, they do

offer much promise (Ramachandran et al., 2005). Go to: Applications of protein chips

The biochemistries of thousands of proteins can be characterized and quantified in a parallel format through the use of protein microarrays. Not only have protein chips been

used to characterize the functions of previously uncharacterized proteins, they have also

 been used to discover new functionalities for previously characterized proteins. Proteome

chips have been used to study protein-protein interactions (Zhu et al., 2001), protein-DNA interactions (Hall et al., 2004), protein-lipid interactions (Zhu et al., 2001), protein-

drug interactions (Huang et al., 2004), protein-receptor interactions (Jones et al., 2006),

and antigen-antibody interactions (Michaud et al., 2003). In addition, proteome chips

have been used to study kinase activities (Ptacek et al., 2005; Zhu et al., 2000) and have been used for serum profiling (Zhu et al., 2006). With respect to protein-protein

interactions, yeast proteome chips have been used to study calmodulin binding proteins

(Zhu et al., 2001). Calmodulin is a calcium binding protein involved in many calcium-regulated cellular pathways (Hook and Means, 2001). Zhu and colleagues biotinylated

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the calmodulin probes, and detected the protein-protein interactions using Cy3-labeled

streptavidin. Their study found 6 known calmodulin binding proteins, and 33 additional

 potential binders. Their study also revealed a novel consensus binding motif that wasrelated to a previously known calmodulin binding motif (Zhu et al., 2001). Hall et al.

used yeast proteome chips to identify previously unrecognized DNA binding activities

(Hall et al., 2004) (Figure 3). Both single and double stranded Cy3 labeled yeast genomicDNA was used to probe a yeast proteome array. Over 200 DNA binding proteins wereidentified, but only about half of those were expected to bind DNA based on their known

function. One of the unexpected targets we found was Arg5,6, a mitochondrial enzyme

involved in arginine biosynthesis. Chromatin immunoprecipitation experiments revealedthat Arg5,6 associates with specific nuclear and mitochondrial loci in vivo. Gel shift

assays revealed that Arg5,6 binds to specific DNA fragments in vitro, and a common

 binding motif was found. Real time PCR experiments with Arg5,6 indicated that it may

have a role in regulating gene expression. Thus, a yeast proteome chip was used todiscover a novel DNA binding protein, and a metabolic enzyme was found that appears

to have a role in directly regulating eukaryotic gene expression. Figure 3 Figure 3

Identification of DNA-binding proteins using a functional protein microarray Zhu andcolleagues also used yeast proteome chips to study phosphoinositide (PI) binding proteins

(Zhu et al., 2001). Phosphoinositides are constituents of cellular membranes and also

regulate a number of diverse cellular processes (Fruman et al., 1998; Odorizzi et al.,

2000). Biotinylated PI liposomes were used to probe the chip. They were detected usingCy-3 labeled streptavidin. Of the 150 novel lipid binding proteins that were identified, 52

corresponded to uncharacterized proteins, and 45 were membrane associated proteins.

Proteome microarrays are also an excellent way to discover drug targets. Entire proteomes printed on a chip can be probed with small molecules in one experiment to

discover interactions. Huang and colleagues used yeast proteome microarrays to study

 protein-drug interactions (Huang et al., 2004). They probed the array with a biotinylated

small molecule inhibitor of rapamycin (SMIR) to find protein targets that may beinvolved in the target-of-rapamycin (TOR) dependent nutrient response network. They

discovered a protein of previously unknown function to be a target of the SMIR. They

followed up the array probing experiment with gene deletion experiments to prove thatthe target protein was indeed a legitimate target of the SMIR. Jones and colleagues have

used protein microarrays to study protein recruitment to receptors in a high-throughput

fashion. Using the data obtained from the microarrays, they also calculated thedissociation constants of the protein-receptor binding (Jones et al., 2006). Specifically,

they created protein microarrays containing most of the human Src homology 2 (SH2)

and phosphotyrosine binding (PTB) domains. These binding domains are known to

interact with different epidermal growth factor receptors (EGFR), which themselves areinvolved in a wide variety of cellular responses. The activated receptors become

 phosphorylated on their tyrosine residues, which then become the sites for the SH2 and

PTB domain binding. The human binding domain microarray of 159 proteins was probed

with 66 fluorescently labeled peptides representing the binding sites of the epidermalgrowth factor receptors. The microarrays were probed with eight concentrations of each

 peptide, and the resulting fluorescence data was used to calculate dissociation constants

to determine the binding affinities of the peptide-binding domain interactions. Theaccuracy of a sample of the dissociation constants calculations was confirmed with

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surface plasmon resonance experiments. From their experimental data, they constructed a

quantitative EGFR interaction map. Their data not only confirmed previously known

interactions, but also showed novel biophysical interactions of SHT2 and PTB domainswith the epidermal growth factor receptor network. With their quantitative data, they

found that different receptor tyrosine kinases differ in their level of selectivity when

overexpressed, which they suggest may be a clue as to why some receptors have moreoncogenic potential than others. There have been multiple yeast protein kinase studiesusing protein chips. Zhu and colleagues used silicone elastomer nanowell sheets placed

onto glass slides to study the activity of 119 yeast kinases using 17 different substrates

(Zhu et al., 2000). The 119 kinases were overexpressed and covalently attached to thenanowell chip. The kinases were incubated with 17 different substrates along with radio

labeled ATP to test for in vitro kinase activity. They obtained many novel results,

including that 27 yeast kinases can act in vitro as tyrosine kinases. This is roughly triple

the number of tyrosine kinases originally thought to exist in yeast. Ptacek and colleagueshave also studied protein phosphorylation in yeast using proteome chips (Ptacek et al.,

2005). Their goal was to develop a global kinase-substrate map for yeast. To this end,

they incubated 87 different yeast protein kinases or kinase complexes separately withradio labeled ATP on a yeast proteome chip containing 4400 unique proteins (Figure 4).

They found about 4200 phosphorylation events affecting 1325 different proteins, and

from this data assembled an in vitro phosphorylation network. Figure 4 Figure 4 A. A

yeast proteome microarray containing ~4,400 GST-tagged yeast proteins printed induplicate. The slide was probed with anti-GST antibodies followed by Cy5-labeled anti-

rabbit antibodies. 40 of 48 blocks are shown. Proteome chips have also been used

successfully to screen patient’s sera for the presence of autoantibodies (Hueber et al.,2005; Kattah et al., 2006) or viral specific antibodies (Zhu et al., 2006). Zhu and

colleagues created a coronavirus protein microarray and used the array to rapidly screen

 patient’s sera for the presence of antibodies against the SARS-CoV coronavirus (Zhu et

al., 2006). They fabricated a protein microarray using coronavirus proteins overexpressedin yeast. The proteins were spotted in duplicate onto microscope slides. Human serum

samples from SARS infected or healthy subjects were screened using the protein chips,

and bound antibodies were detected fluorescently using Cy3-labelled anti-human IgG orIgM antibodies. They found that their coronavirus protein arrays were able to accurately

diagnose the presence of antibodies against coronaviruses in greater than 90% of the

 patient serum samples they tested. Accordingly, the proteome array they created was anexcellent way to quickly diagnose those patients displaying symptoms of SARS infection.

Another set of antigen-antibody experiments using proteome microarrays was performed

 by Michaud and colleagues (Michaud et al., 2003). To analyze antibody specificity, they

screened 11 monoclonal and polyclonal antibodies against a yeast proteome microarraymade up of approximately 5000 different yeast proteins. Fluorescent detection was

 performed using secondary antibodies labeled with Cy5. They found varying degrees of

cross-reactivity among the antibodies that could not necessarily be predicted according to

the amino acid sequences of the proteins in question. Their data should prove useful inevaluating assays using antibodies included in their study. Go to: Conclusion Proteome

chip technology is an excellent high-throughput method used to probe an entire collection

of proteins for a specific function or biochemistry. It is an exceptional new way todiscover previously unknown multifunctional proteins, and to discover new

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functionalities for well-studied proteins. Go to: Acknowledgments We thank Heng Zhu

for comments on the manuscript. Go to: Footnotes Publisher's Disclaimer: This is a PDF

file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will

undergo copyediting, typesetting, and review of the resulting proof before it is published

in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Go to: References Angenendt P, Glokler J, Murphy D, Lehrach H, Cahill

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52. Protein microarray TeknologiDavid A. Hall, Jason Ptacek, dan penulis Michael Snydercorresponding

Penulis informasi ► Copyright dan informasi Lisensi ► 

Versi final penerbit diedit dari artikel ini tersedia di Mech Penuaan DevLihat artikel lainnya di PMC yang mengutip artikel yang diterbitkan.

Pergi ke:

abstrak

Chip protein telah muncul sebagai pendekatan yang menjanjikan untuk berbagai aplikasi

termasuk identifikasi interaksi protein-protein, protein-fosfolipid interaksi, target molekul

kecil, dan substrat protein kinase. Mereka juga dapat digunakan untuk diagnosa klinis dankeadaan penyakit pemantauan. Artikel ini metode saat ini di generasi dan aplikasi

mikroarray protein.

Memahami sistem selular yang kompleks akan membutuhkan identifikasi dan analisis

dari masing-masing komponen dan menentukan bagaimana mereka berfungsi bersama-

sama dan diatur. Sebuah langkah penting dalam proses ini adalah untuk menentukan

kegiatan biokimia protein dan bagaimana kegiatan ini sendiri dikendalikan dandimodifikasi oleh protein lain. Secara tradisional, kegiatan biokimia protein telah

dijelaskan dengan mempelajari molekul tunggal, satu percobaan pada suatu waktu. Proses

ini tidak optimal, karena lambat dan padat karya.

Berbeda dengan pendekatan tradisional ini, tinggi-throughput metode ilmiah telah

dikembangkan dalam dekade terakhir untuk mengoptimalkan studi sejumlah besar

molekul, termasuk DNA, protein dan metabolit. DNA microarray khususnya telahterbukti berharga dalam penelitian genom (Schena et al., 1995). Mereka telah digunakan

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untuk mempelajari pola ekspresi gen, untuk mencari faktor transkripsi situs mengikat,

dan untuk mendeteksi mutasi urutan dan penghapusan dalam skala besar. Namun, DNA

microarray memberitahu kami hanya tentang gen itu sendiri dan memberikan sedikitinformasi mengenai fungsi protein mereka mengkodekan. Baru-baru ini pendekatan

throughput yang tinggi telah dikembangkan untuk studi protein, termasuk profil protein

menggunakan spektrometri massa (Gavin et al, 2006;. Gavin et al, 2002;. Ho et al, 2002;.Krogan et al., 2006; Washburn et al, 2001), penandaan dan lokalisasi subselular(Ghaemmaghami et al, 2003;... Huh et al, 2003) dan microarray protein (MacBeath dan

Schreiber, 2000; Zhu et al, 2001;. Zhu et al, 2000. ). Artikel ini meninjau perkembangan

dalam produksi dan penerapan mikroarray protein.Pergi ke:

Jenis mikroarray protein

Tiga jenis mikroarray protein yang saat ini digunakan untuk mempelajari aktivitas biokimia protein: microarray analitis, microarray fungsional, dan fase microarray

terbalik. Microarray analisis biasanya digunakan untuk profil campuran kompleks protein

dalam rangka untuk mengukur afinitas mengikat, kekhususan, dan tingkat ekspresi protein protein dalam campuran. Dalam teknik ini, sebuah perpustakaan antibodi,

aptamers, atau affibodies tersusun pada slide kaca mikroskop. Array kemudian diperiksa

dengan larutan protein. Mikroarray antibodi adalah microarray analisis yang paling

umum (Bertone dan Snyder, 2005).

Jenis mikroarray dapat digunakan untuk memonitor profil ekspresi diferensial dan

diagnosa klinis. Contoh termasuk respon profiling terhadap stres lingkungan dan sehatdibandingkan jaringan penyakit (Sreekumar et al., 2001).

Fungsional mikroarray protein berbeda dari array analitis dalam array protein fungsional

terdiri dari array yang mengandung protein fungsional full-length atau domain protein.Protein chip ini digunakan untuk mempelajari aktivitas biokimia dari seluruh proteoma

dalam percobaan tunggal. Mereka digunakan untuk mempelajari berbagai interaksi

 protein, seperti protein-protein, protein-DNA, protein-RNA, protein-fosfolipid, dan protein-kecil interaksi molekul (Hall et al, 2004;. Zhu et al, 2001.).

Jenis ketiga microarray protein, terkait dengan microarray analisis, dikenal sebagai faseterbalik microarray protein (RPA). Di RPA, sel terisolasi dari berbagai jaringan yang

menarik dan segaris. Lisat yang tersusun ke slide nitroselulosa menggunakan kontak pin

microarrayer. Slide kemudian diperiksa dengan antibodi terhadap protein target bunga,

dan antibodi biasanya terdeteksi dengan chemiluminescent, neon, atau tes kolorimetri.Peptida referensi yang tercetak pada slide untuk memungkinkan kuantifikasi protein dari

lisat sampel.

RPAs memungkinkan untuk penentuan adanya protein diubah yang mungkin hasil dari penyakit. Secara khusus, modifikasi pasca-translasi, yang biasanya diubah sebagai akibat

dari penyakit, dapat dideteksi dengan menggunakan RPAs (Speer et al., 2005). Setelah

ditentukan yang protein jalur mungkin disfungsional dalam sel, terapi tertentu dapatditentukan untuk menargetkan jalur protein disfungsional dan mengobati penyakit yang

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menarik.

Pergi ke:

 perpustakaan proteome

Tantangan untuk menciptakan proteoma microarray tidak hanya mencakup penciptaan

diperlukan perpustakaan over-ekspresi, tetapi juga pengembangan ekspresi tinggi-throughput dan protokol pemurnian diperlukan untuk secara efisien menghasilkan ribuanmurni, protein fungsional yang dapat bergerak ke permukaan padat . Ada dua strategi

rekombinasi kloning utama yang digunakan untuk membuat perpustakaan frame baca

terbuka (ORFs) dalam vektor ekspresi (Phizicky et al., 2003). Yang pertamamemanfaatkan rekombinasi dalam ragi. Frame terbuka pembacaan bunga diperkuat

sehingga mereka semua mengandung umum 5 'dan berbeda umum 3' berakhir. Para ORFs

dicampur dengan vektor linier yang berisi berakhir identik dengan 5 'dan 3' berakhir dari

ORFs diperkuat. Campuran ini kemudian berubah menjadi ragi di mana kesenjangan-dimediasi rekombinasi terjadi (Phizicky et al., 2003). Koleksi ragi proteoma pertama

diproduksi dengan cara ini (Zhu et al., 2001).

Strategi kloning utama lainnya memanfaatkan Gateway sistem kloning rekombinasi

(Phizicky et al., 2003). Sistem Gateway mengambil keuntungan dari integrasi dan eksisi

sifat fag λ di E. coli. λ fag terintegrasi ke host yang DNA oleh rekombinasi antara situs P

λ att dan situs host att B. Hasil rekombinasi DNA dengan ujung yang disebut att L dan attR. Setelah eksisi fag λ, rekombinasi antara att L dan hasil att R di att P dan att B situs

asli. Sistem Gateway mengambil keuntungan dari sifat unik dari λ fag untuk menciptakan

vektor ekspresi dengan ORF menarik melalui reaksi rekombinasi 2 langkah. Pertama,komponen integrasi λ dicampur dengan ORF diperkuat yang berisi 2 situs B directional

att berbeda. Campuran ini kemudian digabungkan, in vitro, dengan vektor yang berisi

sesuai situs P att. Rekombinasi terjadi dan vektor yang dihasilkan kemudian berisi dua att

L yang berbeda. Vektor ini kemudian dikombinasikan dengan vektor ekspresi yang berisiatt R yang sesuai. Rekombinasi terjadi in vitro, dan ORF dari bunga maka dalam vektor

ekspresi yang berguna. Sistem Gateway cukup fleksibel karena memungkinkan untuk

memudahkan bolak dari ORFs antara vektor ekspresi yang berbeda. Sistem Gatewaytelah digunakan untuk membuat ORF koleksi gen dari ragi (Gelperin et al., 2005), C.

elegans (Reboul et al., 2003), dan manusia (Rual et al., 2004b).

Generasi perpustakaan proteome pertama dan penggunaannya dalam microarray

dilakukan untuk ragi. Perpustakaan pertama mengandung lebih dari 5800 protein ragi,

yang ditandai pada mereka N-terminus dengan GST-HisX6 untuk kemudahan pemurnian.

Setelah mengungkapkan dan memurnikan protein dalam mode high-throughput dalamragi, protein yang terlihat ke slide kaca (Zhu et al., 2001). Sinyal protein pada chip yang

diukur dengan menyelidiki tag GST-HisX6 dengan antibodi anti-GST, diikuti dengan

inkubasi dengan antibodi sekunder fluorophore-terkonjugasi. Protein-protein dan protein-

lipid percobaan yang dilakukan pada chip menyebabkan penemuan protein yangmengikat kalmodulin dan fosfolipid baru dan bukti bahwa protein bergerak pada array

memang aktif.

Baru-baru ini, Gelperin dan rekan telah menciptakan sebuah perpustakaan proteoma ragi

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C-terminal protein TAP-tag yang memungkinkan untuk pemurnian lebih efisien

transmembran dan protein disekresikan (Gelperin et al., 2005). Koleksi Gelperin dan

kolega memanfaatkan teknologi rekombinasi Gateway, yang memungkinkan untukkemudahan transfer ORFs menjadi berbagai vektor ekspresi.

Selain koleksi proteoma dalam ragi yang ada, upaya seperti proyek ORFeome sedangdilakukan untuk membuat koleksi klon pada eukariota lebih tinggi (Brasch et al, 2004;..Reboul et al, 2003;. Rual et al, 2004a; Rual et al. , 2004b). Vidal dan rekan menentukan

ORFeome sebagai set encoding protein frame pembacaan terbuka untuk seluruh

organisme. Mereka telah menggunakan teknologi kloning Gateway rekombinasi (Hartleyet al., 2000) untuk membuat pertama C. elegans ORFeome (Reboul et al., 2003), dan

versi pertama dari ORFeome manusia (Rual et al., 2004b). Koleksi ORFeome manusia

dan microarray juga telah dihasilkan oleh Invitrogen. ORFeomes ini sangat membantu

dalam meningkatkan penjelasan dari genom, dan karena mereka dibuat denganmenggunakan teknologi Gateway, mereka ada dalam format yang fleksibel yang

memungkinkan untuk ekspresi tinggi-throughput protein dalam sejumlah sistem

eksperimental yang berbeda.

Sukses kloning ORF pada eukariota lebih tinggi tergantung pada koleksi cDNA panjang

 penuh, dan ada banyak upaya publik dilakukan untuk membuat koleksi panjang cDNA

manusia penuh, seperti set Unigene (Wheeler et al., 2004), Expression Full Length(FLEX) repositori gen (Brizuela et al., 2001), Analisis Terpadu Molekuler genom dan

Ekspresi (IMAGE) koleksi cDNA mereka (Lennon dkk., 1996), dan mamalia Gene

Koleksi (MGC) (Gerhard et al., 2004) . Koleksi komersial juga tersedia dari perusahaanseperti Invitrogen, GeneCopoeia, OriGene, dan Open Biosystems.

Sebuah microarray protein hanya berguna sebagai kualitas protein tersusun pada chip.

Produksi protein dalam sistem homolog (yaitu protein ragi dinyatakan dalam ragi)diharapkan untuk lebih meningkatkan kualitas protein dan menghasilkan mereka dalam

keadaan aktif. Karena fungsi dari banyak jika tidak sebagian besar protein tidak

diketahui, tidak mungkin untuk benar-benar menentukan apakah setiap protein pada arrayaktif dan fungsional. Sangat mungkin, bagaimanapun, bahwa untuk sebagian besar

 protein pada ragi proteome chip setidaknya beberapa bahan fungsional, karena sejumlah

kegiatan biokimia yang sukses telah dikonfirmasi dan ditemukan menggunakan chip proteoma kami. Kegiatan biokimia meliputi protein-protein, protein-fosfolipid, protein-

DNA, dan protein-kecil interaksi molekul (Hall et al, 2004;. Huang et al, 2004;.. Zhu et

al, 2001), serta reaksi enzimatik (Ptacek et al, 2005;.. Zhu et al, 2000) (Gambar 1).

Gambar 1Gambar 1

Aplikasi mikroarray protein fungsional

Ada kemungkinan bahwa posisi tag afinitas yang digunakan untuk pemurnian dapatmengganggu fungsi dari protein. Penggunaan dua koleksi ragi proteome, satu dengan tag

C-terminal, dan satu dengan tag N-terminal yang berbeda diharapkan dapat saling

melengkapi (Gelperin et al, 2005;. Zhu et al, 2001.). Jika salah satu versi dari proteinditandai menunjukkan hilangnya fungsi akibat lokasi tag, maka versi tagged lain dari

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 protein dapat digunakan pada chip.

Baru-baru ini sebuah metode telah dijelaskan untuk produksi langsung dari protein padachip. DNA adalah melihat pada slide mikroskop dan dikenai transkripsi dan translasi

sistem in vitro. Protein diproduksi sebagai fusi GST dan mematuhi glutathione pada

 permukaan slide. Menggunakan sistem ini LaBaer dan rekan kerja telah menghasilkansejumlah protein manusia yang terlibat dalam metabolisme DNA dan menunjukkaninteraksi protein-protein (Ramachandran et al., 2004). Metode ini menguntungkan karena

menghasilkan protein secara langsung pada slide tanpa memerlukan pemurnian dan

 protein tidak perlu disimpan.Pergi ke:

chip protein

Biasanya, chip protein disusun oleh melumpuhkan protein ke kaca obyek yangdiperlakukan menggunakan spotter kontak (MacBeath dan Schreiber, 2000; Zhu et al,

2001.) Atau microarrayer non-kontak (Delehanty, 2004; Delehanty dan Ligler, 2003;

Jones et al., 2006). Sangat penting bahwa protein tetap berada di lingkungan yang basah.Dengan demikian, buffer sampel mengandung persen tinggi gliserol, dan proses

 pencetakan dilakukan dalam kelembaban dikontrol lingkungan (MacBeath dan Schreiber,

2000;. Zhu et al, 2001). Karena peralatan dan prosedur yang dikembangkan untuk DNA

microarray yang mudah beradaptasi dengan perkembangan mikroarray protein, pilihanmenggunakan slide mikroskop diperlakukan mengikuti dari tersedianya arrayers robot

dan scanner laser yang telah menjadi biasa dalam dunia DNA microarray (Bertone dan

Snyder 2005).

Sejumlah permukaan slide yang berbeda dapat digunakan untuk chip protein. Dalam

memilih permukaan geser, tujuan harus melumpuhkan protein pada chip, menjaga

konformasi dan fungsi protein, dan mencapai kapasitas pengikatan maksimum (Zhu et al.,2003). Hal ini juga penting untuk mempertimbangkan apakah acak atau orientasi seragam

 protein pada permukaan geser yang diinginkan (Gambar 2). Untuk lampiran acak protein

melalui amina, permukaan kaca aldehyde- dan epoxy-diderivatisasi dapat digunakan(Kusnezow et al, 2003;. MacBeath dan Schreiber, 2000). Melapisi permukaan kaca

dengan nitroselulosa, bantalan gel, atau poli-L-lisin juga mencapai orientasi acak protein

sebagai protein secara pasif teradsorpsi ke permukaan (Angenendt et al, 2002;. Charles etal, 2004;. Kramer et al, 2004;. Stillman dan Tonkinson, 2000;. Zhu et al, 2003).

Gambar 2

Gambar 2

Metode lampiran Protein

Permukaan tag Affinity dapat digunakan untuk orientasi seragam protein pada permukaan

keping. Salah satu pilihan geser populer adalah nikel dilapisi slide untuk digunakan

dengan HisX6 tagged protein (Zhu et al., 2003). Lain streptavidin slide dilapisi(Lesaicherre et al., 2002). Tampilan protein dari permukaan chip yang harus memberikan

reagen akses yang lebih mudah ke situs aktif dari protein (Zhu et al., 2003).

Akhirnya microwell juga dapat digunakan untuk tes protein. Zhu dan rekannya

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mengembangkan permukaan geser yang berisi microwell terbuat dari elastomer silikon

dan melekat ke protein microwell epoxy-diobati (Zhu et al., 2000). Manfaat microwell

adalah kemampuan untuk melakukan eksperimen dalam lingkungan berair sementaramencegah kontaminasi silang.

Pergi ke:

metode pendeteksian

Untuk menemukan protein reaktif pada chip proteome, probe molekul kecil diberi label

dengan tag baik neon, afinitas, fotokimia, atau radioisotop. Label neon umumnya lebih

disukai, karena mereka aman dan efektif dan kompatibel dengan tersedia scannermicroarray laser. Namun, probe dapat juga diberi label dengan tag afinitas atau tag

fotokimia (Colca dan Harrigan, 2004; Mitsopoulos et al, 2004.). Huang dan rekan-

rekannya menggunakan biotin berlabel molekul kecil untuk menyelidiki chip proteoma

untuk inhibitor molekul kecil rapamycin (Huang et al., 2004). Protein reaktifdiidentifikasi dengan Cy3 berlabel streptavidin.

Terlepas dari jenis label yang digunakan, ada masalah yang terkait dengan label molekulyang digunakan untuk menyelidiki chip proteoma. Kepala di antara masalah ini adalah

kemungkinan bahwa label itu sendiri dapat mengganggu kemampuan probe untuk

 berinteraksi dengan protein target. Untuk mengatasi masalah ini, beberapa metode deteksi

 bebas label baru-baru ini dikembangkan. Metode deteksi Label-bebas tidak hanyamengatasi masalah halangan sterik dari label, tetapi juga memungkinkan untuk

 pengumpulan data mengikat kinetik (ditinjau dalam (Ramachandran et al., 2005)).

Teknologi terkemuka saat ini untuk deteksi bebas label interaksi protein plasmon

resonansi permukaan (SPR), yang probe indeks lokal bias. Pilihan lain termasuk

nanotube karbon, kawat nano karbon, dan sistem microelectromechanical cantilevers.

Sementara teknologi ini masih dalam tahap awal dan tidak cocok untuk tinggi-throughputdeteksi interaksi protein, mereka menawarkan banyak janji (Ramachandran et al., 2005).

Pergi ke:

Aplikasi chip protein

Para biochemistries ribu protein dapat dicirikan dan diukur dalam format paralel melalui

 penggunaan mikroarray protein. Tidak hanya chip protein telah digunakan untukmengkarakterisasi fungsi protein yang sebelumnya uncharacterized, mereka juga telah

digunakan untuk menemukan fungsi baru untuk protein ditandai sebelumnya. Chip

Proteome telah digunakan untuk mempelajari interaksi protein-protein (Zhu et al., 2001),

interaksi protein-DNA (Hall et al., 2004), protein-lipid interaksi (2001 Zhu et al.),Interaksi protein-obat (Huang et al., 2004), interaksi protein-reseptor (Jones et al., 2006),

dan interaksi antigen-antibodi (Michaud et al., 2003). Selain itu, chip proteoma telah

digunakan untuk mempelajari aktivitas kinase (Ptacek et al, 2005;.. Zhu et al, 2000) dan

telah digunakan untuk serum profiling (Zhu et al, 2006.).

Sehubungan dengan interaksi protein-protein, chip ragi proteoma telah digunakan untuk

mempelajari protein yang mengikat kalmodulin (Zhu et al., 2001). Kalmodulin adalah protein yang mengikat kalsium terlibat dalam banyak jalur seluler kalsium diatur (Hook

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and Means, 2001). Zhu dan rekan biotin probe kalmodulin, dan mendeteksi interaksi

 protein-protein menggunakan Cy3-berlabel streptavidin. Studi mereka menemukan 6

dikenal kalmodulin mengikat protein, dan 33 pengikat potensi tambahan. Studi mereka juga mengungkapkan novel konsensus yang mengikat motif yang terkait dengan

kalmodulin mengikat dikenal sebelumnya motif (Zhu et al., 2001).

Balai et al. digunakan chip ragi proteoma untuk mengidentifikasi kegiatan mengikatDNA yang sebelumnya tidak dikenal (Hall et al., 2004) (Gambar 3). Kedua Cy3 berlabel

ragi DNA genom tunggal dan double stranded digunakan untuk menyelidiki array ragi

 proteoma. Lebih dari 200 protein DNA mengikat diidentifikasi, tetapi hanya sekitarsetengah dari mereka diperkirakan untuk mengikat DNA berdasarkan fungsi diketahui

mereka. Salah satu target yang tak terduga yang kami temukan adalah Arg5,6, enzim

mitokondria yang terlibat dalam biosintesis arginin. Percobaan immunoprecipitation

kromatin mengungkapkan bahwa rekan Arg5,6 dengan nuklir spesifik dan lokusmitokondria in vivo. Tes pergeseran Gel mengungkapkan bahwa Arg5,6 mengikat

fragmen DNA spesifik in vitro, dan motif yang mengikat umum ditemukan. Real time

PCR eksperimen dengan Arg5,6 menunjukkan bahwa hal itu mungkin memiliki perandalam mengatur ekspresi gen. Dengan demikian, chip ragi proteoma digunakan untuk

menemukan DNA baru binding protein, dan enzim metabolik ditemukan bahwa

tampaknya memiliki peran dalam mengatur ekspresi gen secara langsung eukariotik.

Gambar 3Gambar 3

Identifikasi protein DNA-binding menggunakan protein microarray fungsional

Zhu dan rekannya juga menggunakan chip ragi proteoma untuk mempelajari

 phosphoinositide (PI) mengikat protein (Zhu et al., 2001). Phosphoinositides adalah

konstituen dari membran sel dan juga mengatur sejumlah proses seluler yang beragam

(Fruman et al, 1998;.. Odorizzi et al, 2000). Liposom PI terbiotinilasi digunakan untukmenyelidiki chip. Mereka terdeteksi menggunakan Cy-3 berlabel streptavidin. Dari 150

 protein yang mengikat lipid baru yang diidentifikasi, 52 berhubungan dengan protein

uncharacterized, dan 45 adalah membran protein terkait.

Microarray Proteome juga merupakan cara yang baik untuk menemukan target obat.

Proteomes Seluruh dicetak pada sebuah chip bisa dideteksi dengan molekul kecil dalamsatu percobaan untuk menemukan interaksi. Huang dan rekan-rekannya menggunakan

ragi microarray proteoma untuk mempelajari interaksi protein-obat (Huang et al., 2004).

Mereka diperiksa array dengan inhibitor molekul terbiotinilasi kecil rapamycin (SMIR)

untuk menemukan target protein yang mungkin terlibat dalam (TOR) jaringan respon gizisasaran-of-rapamycin tergantung. Mereka menemukan protein fungsi yang sebelumnya

tidak diketahui menjadi target SMIR. Mereka ditindaklanjuti array menyelidik percobaan

dengan percobaan penghapusan gen untuk membuktikan bahwa protein target itu

memang target yang sah dari SMIR.

Jones dan rekan telah menggunakan microarray protein untuk mempelajari perekrutan

 protein reseptor secara high-throughput. Menggunakan data yang diperoleh darimicroarray, mereka juga menghitung konstanta disosiasi protein reseptor mengikat (Jones

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et al., 2006). Secara khusus, mereka menciptakan microarray protein yang mengandung

sebagian besar manusia Src homologi 2 (SH2) dan phosphotyrosine (PTB) domain yang

mengikat. Domain mengikat ini diketahui berinteraksi dengan berbagai faktor pertumbuhan epidermal reseptor (EGFR), yang sendiri terlibat dalam berbagai respon

seluler. Reseptor yang diaktifkan menjadi terfosforilasi pada residu tirosin mereka, yang

kemudian menjadi situs untuk domain SH2 dan PTB mengikat. The microarray domainmengikat manusia dari 159 protein yang diperiksa dengan 66 peptida fluorescent berlabelmewakili situs pengikatan faktor pertumbuhan epidermal reseptor. Mikroarray diperiksa

dengan delapan konsentrasi masing-masing peptida, dan data fluoresensi yang dihasilkan

digunakan untuk menghitung konstanta disosiasi untuk menentukan afinitas pengikataninteraksi domain peptida mengikat. Keakuratan sampel perhitungan konstanta disosiasi

dikonfirmasi dengan plasmon permukaan percobaan resonansi. Dari data eksperimen

mereka, mereka membangun sebuah peta interaksi EGFR kuantitatif. Data mereka tidak

hanya dikonfirmasi interaksi dikenal sebelumnya, tetapi juga menunjukkan interaksi biofisik novel SHT2 dan PTB domain dengan jaringan reseptor faktor pertumbuhan

epidermal. Dengan data kuantitatif, mereka menemukan bahwa reseptor tirosin kinase

yang berbeda berbeda dalam tingkat selektivitas ketika diekspresikan, yang merekamenyarankan mungkin merupakan petunjuk mengapa beberapa reseptor memiliki potensi

lebih onkogenik daripada yang lain.

Ada beberapa studi protein kinase ragi menggunakan chip protein. Zhu dan rekan-rekannya menggunakan silikon lembaran elastomer nanowell ditempatkan ke slide kaca

untuk mempelajari aktivitas 119 kinase ragi menggunakan 17 substrat yang berbeda (Zhu

et al., 2000). 119 kinase yang diekspresikan dan kovalen melekat chip nanowell. Kinasediinkubasi dengan 17 substrat yang berbeda bersama dengan radio berlabel ATP untuk

menguji in vitro aktivitas kinase. Mereka memperoleh banyak hasil baru, termasuk yang

27 kinase ragi dapat bertindak secara in vitro sebagai kinase tirosin. Ini kira-kira tiga kali

lipat jumlah kinase tirosin yang diperkirakan ada dalam ragi.

Ptacek dan rekannya juga telah mempelajari fosforilasi protein dalam ragi menggunakan

chip proteoma (Ptacek et al., 2005). Tujuan mereka adalah untuk mengembangkan petakinase-substrat global untuk ragi. Untuk tujuan ini, mereka diinkubasi 87 protein kinase

ragi yang berbeda atau kompleks kinase secara terpisah dengan radio berlabel ATP pada

chip ragi proteoma mengandung protein yang unik 4400 (Gambar 4). Merekamenemukan sekitar 4200 peristiwa fosforilasi mempengaruhi 1.325 protein yang berbeda,

dan dari data ini dirakit sebuah vitro jaringan fosforilasi di.

Gambar 4

Gambar 4A. Sebuah ragi proteoma microarray mengandung ~ 4.400 protein ragi GST-tag dicetak

dalam rangkap dua. Slide itu diperiksa dengan antibodi anti-GST diikuti oleh Cy5-label

antibodi anti-kelinci. 40 dari 48 blok yang akan ditampilkan.

Chip Proteome juga telah berhasil digunakan untuk sera layar pasien untuk adanya

autoantibodi (Hueber et al, 2005;. Kattah et al, 2006.) Atau antibodi spesifik virus (Zhu et

al, 2006.). Zhu dan rekan menciptakan microarray protein coronavirus dan digunakanarray dengan cepat layar sera pasien untuk adanya antibodi terhadap coronavirus SARS-

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CoV (Zhu et al., 2006). Mereka mengarang microarray protein menggunakan protein

coronavirus diekspresikan dalam ragi. Protein yang terlihat dalam rangkap ke slide

mikroskop. Sampel serum manusia dari subyek yang terinfeksi atau sehat SARS disaringmenggunakan chip protein, dan antibodi terikat terdeteksi fluorescent menggunakan Cy3-

 berlabel IgG atau IgM antibodi anti-manusia. Mereka menemukan bahwa coronavirus

mereka protein array mampu untuk secara akurat mendiagnosis adanya antibodi terhadapcoronavirus di lebih dari 90% dari sampel serum pasien mereka diuji. Oleh karena itu,array proteome mereka ciptakan adalah cara terbaik untuk cepat mendiagnosa pasien

menunjukkan gejala-gejala infeksi SARS.

Set percobaan antigen-antibodi menggunakan microarray proteoma dilakukan oleh

Michaud dan rekan (Michaud et al., 2003). Untuk menganalisis spesifisitas antibodi,

mereka disaring 11 monoklonal dan poliklonal antibodi terhadap ragi proteoma

microarray terdiri dari sekitar 5000 protein ragi yang berbeda. Deteksi Fluorescentdilakukan dengan menggunakan antibodi sekunder berlabel Cy5. Mereka menemukan

 berbagai tingkat reaktivitas silang antara antibodi yang tidak bisa selalu diprediksi sesuai

dengan urutan asam amino dari protein yang bersangkutan. Data mereka harusmembuktikan berguna dalam mengevaluasi tes menggunakan antibodi yang termasuk

dalam studi mereka.

Pergi ke:

kesimpulan

Teknologi proteoma chip metode tinggi-throughput sangat baik digunakan untuk

menyelidiki seluruh koleksi protein untuk fungsi atau biokimia tertentu. Ini adalah cara baru yang luar biasa untuk menemukan protein multifungsi yang sebelumnya tidak

diketahui, dan untuk menemukan fungsi baru untuk protein dipelajari dengan baik.

Pergi ke:

Ucapan Terima Kasih

Kami berterima kasih kepada Heng Zhu untuk komentar pada naskah.

Pergi ke:Catatan kaki

Penerbit Disclaimer: Ini adalah file PDF dari naskah diedit yang telah diterima untuk publikasi. Sebagai layanan kepada pelanggan kami, kami menyediakan versi awal ini

naskah. Naskah akan menjalani copyediting, typesetting, dan review bukti yang

dihasilkan sebelum diterbitkan dalam bentuk citable akhir. Harap dicatat bahwa selama

kesalahan proses produksi dapat ditemukan yang dapat mempengaruhi isi, dan semua penolakan hukum yang berlaku untuk jurnal berhubungan.

Pergi ke:

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Products - Protein Microarrays 

Arrayit sets the standard in the microarray industry with ArrayIt® Brand ProteinMicroarrays. Please review our product list below, and click on a product for protocols andpricing. Click here for scientific publications using Arrayit protein microarrays.

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HuProt™ v2.0 19K Human Proteome Microarrays  

Arrayit HuProt™ v2.0 19K Human Proteome Microarrayscontain 19,275 human proteins representing most of the

human proteome and 119 mouse proteins for total 19,394

proteins. All major protein functional families are representedfor protein-protein, small molecule-protein, antibody binding

specificity, immune profiling, DNA and RNA binding studies ona proteomic scale, advanced contact printing technology and

proprietary surface chemistry, cold room manufacturing andlinear drive robotics, universal 25 x 76 mm glass substrate

slide format, 1 HuProt™ v2.0 19K microarray per kit. 

Human Allergy Test  

Arrayit protein microarray allergy tests includingenvironmental, food and herbal medicine allergens printed as

protein microarrays on standard glass substrate slides for IgE

(123 allergens) and IgG (101 allergens) testing in yourlaboratory. These 1 x 25 x 76 mm microarray slide tests arecompatible with all glass slide-based microarray scanners and

other microarray equipment, pricing is per microarray, forresearch use only.

AllerSpot™ Companion Animal Testing Service  

Arrayit AllerSpot™ Companion Animal Testing Service using a62-allergen microarray based on rigorous scientific studies

and environmental research on various factors affecting the

prevalence rates of allergies. Based on this information, wehave identified a test for the most common allergens inhaledand ingested by our companion animal pets including dogs

and cats. Ask your veterinarian about AllerSpot™. 

Pathogen Antigen Microarrays  

Arrayit Antigen Microarrays, manufactured with Arrayit’spatented microarray manufacturing technology 6,101,946,

are designed for reactive antibody screening, immuneresponse profiling and biomarker research. Each microarray

contains antigens and extensive controls for reactions with 1µl of human serum or plasma immunology, infectious

disease, microbiology, and cell biology. For research use only. 

PlasmaScan Antibody Microarrays  

The only microarray on the market containing a library of

monoclonal antibodies raised against native human plasmaproteins. Native human proteins guarantee proper folding and

native post-translation modification to ensure high antibody

binding affinity and specificity. Use Arrayit PlasmaScan 380 toexplore the human plasma proteome with unprecedentedprecision. Identify new biomarkers in human disease

samples.

Protein Microarray Manufacturing 

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Arrayit Services empowers the production and analysis of a

any protein microarray including proteome microarrays.Proteins are printed into microarrays using our high-speedrobotics onto any surface of interest depending on the

application and detection method. The printed proteins react

with proteins, antibodies, drugs and receptors in solution thatare incubated to the microarray. These protein microarraysare compatible with any microarray detection technique

including standard fluorescence, SPR, chemiluminescence,colorimetric, RLS, mass spec and others.

Tissue Microarrays 

Arrayit Tissue Microarrays (TMAs) contain sectioned tissue

cores printed on glass slides. The diameter of each tissuemicroarray spot is 0.6-2.0 mm and the sections number from10-100+. Human tissue types include 1,972 normal,

malignant and metastatic sources. TMA technology can be

used to rapidly and cost-effectively identify DNA, mRNA andprotein biomarkers for a wide range of disease states and

therapeutic applications.

Transcription Factors  

Microarray transcription factor profiling systems enable

researchers to rapidly identify transcription factors that drive

biological responses. They employ a powerful multiplexedapproach for simultaneously measuring the binding of

different transcription factors to their unique regulatory DNA

elements.

Produk - Mikroarray Protein

Arrayit menetapkan standar dalam industri microarray dengan ArrayIt® Merek Protein Mikroarray.

Silahkan lihat daftar produk kami di bawah ini, dan klik pada produk untuk protokol dan harga. Klik di

sini untuk publikasi ilmiah menggunakan Arrayit protein microarray.

HuProt™ v2.0 19K Human Proteome Mikroarray

proteoma manusia

Arrayit HuProt™ v2.0 19K Human Proteome Mikroarray mengandung 19.275 protein manusia yang

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mewakili sebagian besar proteome manusia dan 119 protein tikus total 19.394 protein. Semua keluarga

fungsional protein utama yang diwakili untuk protein-protein, kecil molekul protein, spesifisitas

pengikatan antibodi, profil kekebalan tubuh, DNA dan RNA mengikat studi pada skala proteomik,

teknologi cetak kontak canggih dan kimia permukaan proprietary, ruang manufaktur dingin dan robotika

berkendara linear , 25 x 76 mm Format geser substrat kaca universal, 1 HuProt™ v2.0 19K microarray

per kit.

Manusia Tes Alergi

tes alergi manusia

Arrayit microarray protein tes alergi termasuk lingkungan, makanan dan alergen jamu dicetak sebagai

microarray protein pada standar slide substrat kaca untuk IgE (123 alergen) dan IgG (101 alergen)

pengujian di laboratorium Anda. Ini 1 x 25 x 76 mm tes microarray geser yang kompatibel dengan semua

kaca berbasis slide scanner microarray dan peralatan microarray lainnya, harga adalah per microarray,

untuk digunakan penelitian saja.

AllerSpot™ Companion Animal Testing Service

alergen-array

Arrayit AllerSpot™ Companion Animal Testing Service menggunakan 62-alergen microarray berdasarkan

studi ilmiah yang ketat dan penelitian lingkungan pada berbagai faktor yang mempengaruhi tingkat

prevalensi alergi. Berdasarkan informasi ini, kami telah mengidentifikasi tes untuk alergen yang paling

umum terhirup dan tertelan oleh hewan peliharaan pendamping hewan termasuk anjing dan kucing.

Tanyakan dokter hewan Anda tentang AllerSpot™.

Patogen Antigen Mikroarray

microarray antigen

Arrayit Antigen Mikroarray, diproduksi dengan teknologi manufaktur dipatenkan microarray Arrayit itu

6.101.946, yang dirancang untuk pemeriksaan antibodi reaktif, profil respon imun dan penelitian

biomarker. Setiap microarray mengandung antigen dan kontrol yang luas untuk reaksi dengan 1 ml

serum manusia atau imunologi plasma, penyakit menular, mikrobiologi, dan biologi sel. Untuk penelitian

hanya menggunakan.

PlasmaScan Antibodi Mikroarray

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Satu-satunya microarray di pasar mengandung perpustakaan antibodi monoklonal yang diajukan

terhadap protein plasma asli manusia. Protein manusia asli menjamin lipat yang tepat dan modifikasi

pasca-translasi asli untuk memastikan afinitas pengikatan antibodi yang tinggi dan spesifisitas. Gunakan

Arrayit PlasmaScan 380 untuk mengeksplorasi plasma proteome manusia dengan presisi belum pernah

terjadi sebelumnya. Mengidentifikasi biomarker baru dalam sampel penyakit manusia.

Protein microarray Manufacturing

Arrayit Layanan memberdayakan produksi dan analisis microarray protein apapun termasuk microarray

proteoma. Protein dicetak ke microarray menggunakan robot berkecepatan tinggi ke permukaan bunga

tergantung pada metode aplikasi dan deteksi. Protein yang dicetak bereaksi dengan protein, antibodi,

obat-obatan dan reseptor dalam larutan yang diinkubasi dengan microarray. Mikroarray protein ini

kompatibel dengan teknik deteksi microarray termasuk fluoresensi standar, SPR, chemiluminescence,

kolorimetri, RLS, spesifikasi massa dan lain-lain.

Mikroarray jaringan

Mikroarray Arrayit Tissue (TMAs) berisi dipotong core jaringan dicetak pada slide kaca. Diameter setiap

tempat jaringan microarray adalah 0,6-2,0 mm dan jumlah bagian 10-100 +. Jenis jaringan manusia

mencakup 1,972 normal, ganas dan metastasis sumber. Teknologi TMA dapat digunakan untuk cepat

dan biaya-efektif mengidentifikasi DNA, mRNA dan protein biomarker untuk berbagai kondisi penyakit

dan aplikasi terapi.

Faktor transkripsi

Microarray faktor transkripsi profil sistem memungkinkan para peneliti untuk secara cepat

mengidentifikasi faktor-faktor transkripsi yang mendorong respons biologis. Mereka menggunakan

pendekatan multiplexing yang kuat untuk secara bersamaan mengukur pengikatan faktor transkripsi

yang berbeda untuk unsur DNA peraturan yang unik.