Protein–protein interaction testing in the yeast two-hybrid assay
Protein–protein interaction testing service (Cat No CT-100)
Attempting to show/confirm binding between two proteins/protein fragments of interest in the yeast two-hybrid (Y2H) system/assay[1,2], which is a well-known technique that can be used to study protein–protein interactions (PPIs). Once a PPI has been confirmed in the Y2H system, involved binding sites can be mapped relatively easily. Once a binding site has been mapped, peptide protein–protein interaction inhibitor candidates for that PPI can be generated.
Yeast two-hybrid assay
The yeast two-hybrid assay can be used to demonstrate physical binding between two proteins of interest experimentally. After necessary plasmids have been constructed (Table 1a), cells of a yeast reporter strain are cotransformed with a specific combination of plasmids (Table 1b). Successfully cotransformed cells (cotransformants, which are new yeast strains) are able to grow on SD-Leu/-Trp Agar plates. Reporter activity (Table 1b) in these new yeast strains can be measured qualitatively and quantitatively.
|Table 1. Protein–protein interaction (PPI) between hypothetical Protein 1 (PRTN1) (1-500) and hypothetical Protein 2 (PRTN2) (1-600) is tested in the yeast two-hybrid system/assay (hypothetical example). (a) C3 expresses Gal4(1-147) fused to PRTN1(1-500); C4 expresses Gal4(768-881) fused to PRTN2(1-600). (b) A1 is a bait–prey interaction assay; A2 is a prey-dependency control assay; A3 is a bait-dependency control assay.|
|(1) Part of Clontech’s Matchmaker® Gold Yeast Two-Hybrid System.|
To measure the activity of, for example, the ADE2 reporter qualitatively: a suspension of cells is placed on SD-Ade/-Leu/-Trp Agar, and if the cells are able to grow (an active ADE2 reporter will give the cells the ability to grow on -Ade minimal medium) a clear patch of cells will become visible after a few days.
To measure the activity of the lacZ reporter quantitatively: from a specific number of cells (grown to mid-log phase) lacZ reporter signal strength is measured.
Physical binding (direct and or indirect) between PRTN1 and PRTN2 is confirmed if assay A1 turns out positive and assays A2 and A3 turn out negative (Table 1b), for explanation, see our Yeast two-hybrid (Y2H) system-webpage.
Estimates/quotes & orders
Currently, this service can be purchased through the Scientist.com platform only. For estimates/quotations, orders and other information see our Protein–Protein Interaction Testing Service (Cat No CT-100) Service Description and our Scientist.com profile. For associated comprehensive (but convenient) legal paperwork, see the example/draft Statement of Work, the Scientist.com Customer Agreement and the Scientist.com Supplier Agreement.
Due to our skills, expertise and highly efficient workflows we can deliver high-quality work at competitive prices.
 Fields, S. & Song, O. (1989), 'A novel genetic system to detect protein-protein interactions.', Nature 340, 245--246.
 Vidal, M. & Fields, S. (2014), 'The yeast two-hybrid assay: still finding connections after 25 years.', Nature methods 11, 1203--1206.
 Development of protein–protein interaction inhibitor candidates
 For details refer to the manual of Clontech’s Matchmaker® Gold Yeast Two-Hybrid System or contact us.
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