Two-hybrid screening

yeast two-hybridyeast two hybridyeast two-hybrid systemyeast two-hybrid screenyeast two-hybrid screeningtwo-hybridyeast 2-hybridyeast two-hybrid assayyeast-two hybridbacterial two-hybrid screen
[[Image:Two hybrid assay.svg|thumb|300px| Overview of two-hybrid assay, checking for interactions between two proteins, called here Bait and Prey.wikipedia
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Stanley Fields (biologist)

Stanley FieldsStan Fields
Pioneered by Stanley Fields and Ok-Kyu Song in 1989, the technique was originally designed to detect protein–protein interactions using the Gal4 transcriptional activator of the yeast Saccharomyces cerevisiae.
Stanley Fields is an American biologist best known for developing the yeast two hybrid method for identifying protein–protein interactions.

Protein-fragment complementation assay

protein-fragment complementation assays
The Y2H is thus a protein-fragment complementation assay.
This principle can be applied to many different reporter proteins and is also the basis for the yeast two-hybrid system, an archetypical PCA assay.

Protein–protein interaction

interactprotein-protein interactionsprotein-protein interaction
Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
The most conventional and widely used high-throughput methods are yeast two-hybrid screening and affinity purification coupled to mass spectrometry.

Protein

proteinsproteinaceousstructural proteins
Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
Key experimental techniques in proteomics include 2D electrophoresis, which allows the separation of many proteins, mass spectrometry, which allows rapid high-throughput identification of proteins and sequencing of peptides (most often after in-gel digestion), protein microarrays, which allow the detection of the relative levels of the various proteins present in a cell, and two-hybrid screening, which allows the systematic exploration of protein–protein interactions.

Gal4 transcription factor

Gal4
Pioneered by Stanley Fields and Ok-Kyu Song in 1989, the technique was originally designed to detect protein–protein interactions using the Gal4 transcriptional activator of the yeast Saccharomyces cerevisiae. A. The Gal4 transcription factor gene produces a two-domain protein (BD and AD) essential for transcription of the reporter gene (LacZ).
The broad use of the Gal4 is in yeast two-hybrid screening to screen or to assay protein-protein interactions in eukaryotic cells from yeast to human.

Saccharomyces cerevisiae

S. cerevisiaeyeastbudding yeast
Pioneered by Stanley Fields and Ok-Kyu Song in 1989, the technique was originally designed to detect protein–protein interactions using the Gal4 transcriptional activator of the yeast Saccharomyces cerevisiae.
These include yeast two-hybrid for studying protein interactions and tetrad analysis.

Reporter gene

reporterreportersassay
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS).
A more complex use of reporter genes on a large scale is in two-hybrid screening, which aims to identify proteins that natively interact with one another in vivo.

High-throughput screening

high throughput screeninghigh-throughputscreening
Millions of potential interactions in several organisms have been screened in the latest decade using high-throughput screening systems (often using robots) and over thousands of interactions have been detected and categorized in databases as BioGRID.

Y187

A number of yeast strains have been created specifically for Y2H screens, e.g. Y187 and AH109, both produced by Clontech.
Y187 is a strain of yeast (Saccharomyces cerevisiae) used in biological research for two-hybrid screening.

AH109

A number of yeast strains have been created specifically for Y2H screens, e.g. Y187 and AH109, both produced by Clontech.
AH109 is a strain of yeast (Saccharomyces cerevisiae) used in biological research for two-hybrid screening.

Lac operon

lac'' operonlacZlac
By placing the hybrid proteins under the control of IPTG-inducible lac promoters, they are expressed only on media supplemented with IPTG.
The lac gene and its derivatives are amenable to use as a reporter gene in a number of bacterial-based selection techniques such as two hybrid analysis, in which the successful binding of a transcriptional activator to a specific promoter sequence must be determined.

DNA-binding protein

DNA binding proteinprotein-DNA interactionDNA-binding
Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively. The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for the activation of transcription.
Yeast one-hybrid System (Y1H) is used to identify which protein binds to a particular DNA fragment.

Phage display

antibody phage display technologyphage display library
Like the two-hybrid system, phage display is used for the high-throughput screening of protein interactions.

Candida albicans

C. albicansCandidayeast
C. albicans is a yeast with a particular feature: it translates the CUG codon into serine rather than leucine.
albicans it is less feasible to use the common host organism (Saccharomyces cerevisiae'') for two-hybrid studies.

Molecular biology

molecular biologistmolecularmolecular microbiology
Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.

DNA

deoxyribonucleic aciddouble-stranded DNAdsDNA
Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.

Upstream and downstream (DNA)

upstreamdownstreamupstream or downstream
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS).

Transcription factor

transcription factorsgene transcription factortranscriptional factors
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS).

Upstream activating sequence

Upstream Activation Sequenceupstream activating sequence (UAS)upstream activator
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS).

Protein domain

domainsdomainstructural domain
The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for the activation of transcription.

Transcription (biology)

transcriptiontranscribedtranscriptional
The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for the activation of transcription.

Escherichia coli

E. coliE.coliE-coli
Since then, the same principle has been adapted to describe many alternative methods, including some that detect protein–DNA interactions or DNA-DNA interactions, as well as methods that use different host organisms such as Escherichia coli or mammalian cells instead of yeast.

Eukaryote

Eukaryotaeukaryoticeukaryotes
The key to the two-hybrid screen is that in most eukaryotic transcription factors, the activating and binding domains are modular and can function in proximity to each other without direct binding.

Genetic engineering

genetically engineeredgenetically modifiedgenetic modification
This system often utilizes a genetically engineered strain of yeast in which the biosynthesis of certain nutrients (usually amino acids or nucleic acids) is lacking.