Yeast two-hybrid assay service: test binary protein–protein interactions

The yeast two-hybrid (Y2H) system / assay (Figure 1) is a well-known technique that can be used to demonstrate physical binding between two proteins of interest experimentally. The system is often very suitable to verify putative / potential protein–protein interactions (PPIs) that were identified using affinity purification-mass spectrometry (AP-MS) or any other method to identify PPIs, including PPI prediction methods. Once a PPI has been confirmed in the Y2H system, involved binding sites can be mapped relatively easily (lean more). Once a binding site has been identified, potential peptide protein–protein interaction inhibitors can be derived (learn more).  See Table 1 to learn about our protein–protein interaction testing service (Cat. No. C200).

 

 

tech yeast two hybrid system fig 1
Figure 1. Protein–protein interaction between retinoblastoma protein and large T antigen protein. (a) Binding between Rb (the ‘bait’ protein) and LTP (the ‘prey’ protein) results in an active reporter gene in cells of the Gal4 yeast two-hybrid (Y2H) system / assay. Data acquired and diagram adapted from NAR 23:1152, Oxford Journals; Rb: Retinoblastoma protein (301-918); LTP (for Large T antigen Peptide): SV40 Large T Antigen (residues 103-115). VMD (http://www.ks.uiuc.edu/Research/vmd/) and coordinates from PDB ID: 1GH6 were used to generate representations of: (b) Retinoblastoma protein (residues 378-772 minus residues 578-644); (c) SV40 Large T Antigen (residues 103-115); and (d) Retinoblastoma protein (residues 378-772 minus residues 578-644) and SV40 Large T Antigen (residues 103-115). Note: for references and further explanation of this Figure and the Y2H system please visit our yeast two-hybrid system web page.

 

 

 

Table 1. Protein–protein interaction testing service (Cat. No. C200). Physical binding (direct and or indirect) between the bait protein and the prey protein is confirmed if the bait–prey interaction / binding assay (Step 2a) turns out positive and the prey-dependency control assay (Step 2b) as well as the bait-dependency control assay (Step 2c) turn out negative, for explanation, see our yeast two-hybrid (Y2H) system web page. To learn more please refer to our PPI Testing Service document.(1)

Step 1a

Bait Plasmid Construction – DNA that encodes the bait (protein of interest (or fragment / mutant form thereof)) will be appropriately cloned as insert into empty bait vector. Starting from 170 euro for a single construct.

Step 1b

Prey Plasmid Construction – DNA that encodes the prey (protein of interest (or fragment / mutant form thereof)) will be appropriately cloned as insert into empty prey vector. Starting from 170 euro for a single construct.

Step 2a

Bait–Prey Binding Assay – cells of a yeast two-hybrid system reporter strain will be cotransformed with the bait plasmid (Step 1a) and the prey plasmid (Step 1b) and subsequently reporter activity in generated cotransformants will measured. Starting from 85 euro for a single assay.

Step 2b

Prey-Dependency Assay – cells of a yeast two-hybrid system reporter strain will be cotransformed with the bait plasmid (Step 1a) and empty prey vector and subsequently reporter activity in generated cotransformants will measured. Starting from 85 euro for a single assay.

Step 2c

Bait-Dependency Assay – cells of a yeast two-hybrid system reporter strain will be cotransformed with empty bait vector and the prey plasmid (Step 1b) and subsequently reporter activity in generated cotransformants will measured. Starting from 85 euro for a single assay.

(1) https://media.cellatechnologies.com/PPI_Testing_Service_C200_v20190627.pdf.
 
 

 

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