Yeast Two-Hybrid basedProtein Interactions screening

Molecules tested: Soluble proteins

• Full-length proteins, fragments or peptides
• Cytoplasmic and secreted proteins
• Loop regions, cytoplasmic tails and extracellular domains of membrane proteins

Key benefits

• Rapid & exhaustive screening of high-complexity domain-enriched libraries
• Identification of weak and rare binding partners
• Up to 380 positive clones sequenced (5' & 3') in a single screen
• Comprehensive & integrated bioinformatics analysis of results and annotation of functional, structural and interacting domains of the interacting proteins
• Back-up screening strategies included
• High-quality scientific & technical assistance regarding bait design and screening strategy for the best possible outcome

Molecules tested: Membrane-associated proteins

• Full-length and fragments of transmembrane-containing proteins

Key benefits

• Identify protein targets of single or multipass membrane proteins
• Functional assays include bait testing for proper localization & orientation
• Rapid & exhaustive screening of custom-made MBmate libraries
• Up to 380 positive clones sequenced
• High-quality scientific & technical assistance regarding bait design and screening strategy for the best possible outcome 

Molecules Tested: DNA sequences (i.e., promoter regions, responsive elements)

Key benefits

• Rapid & exhaustive screening of high-complexity domain-enriched libraries
• Up to 380 positive clones sequenced (5'& 3') in a single screen
• Comprehensive & integrated bioinformatics analysis of results and annotation of functional, structural and interacting domains of the interacting proteins
• Back-up screening strategies included
• High-quality scientific & technical assistance regarding bait design and screening strategy for the best possible outcome

Molecules Tested: RNA sequences

Key Benefits

• Robust MS-2 bait system
• RNA baits can be screened in both 5’ and 3’ orientations
• Up to 380 positive clones sequenced (5'& 3') in a single screen
• Comprehensive & integrated bioinformatics analysis of results and annotation of functional, structural and interacting domains of the interacting proteins
• Project team leader provides scientific & technical assistance regarding bait design and screening strategy for the best possible outcome

The ULTImate Y2H+1 screen is a modified version of the ULTImate Y2H system where the expression of a co-factor in yeast is required for either the proper folding (i.e., chaperone) or post-translational modification (i.e., tyrosine kinase) of the bait. Co-expression of several proteins of a complex is also possible.

Molecule tested: Soluble protein + co-factor 

Key Benefits

• Rapid & exhaustive screening of high-complexity domain-enriched libraries
• Identification of weak and rare binding partners
• Comprehensive & integrated bioinformatics analysis of results and annotation of functional, structural and interacting domains of the interacting proteins
• Project team leader provides scientific & technical assistance regarding bait design and screening strategy for the best possible outcome

Molecules tested: Soluble proteins

• Full-length proteins, fragments or peptides
• Cytoplasmic or extracellular proteins
• Loops and tails of membrane proteins

Key Benefits

• Robust screening method to identify single point mutation(s) that are responsible for the protein-protein interaction (PPI) based on ULTImate Y2H™
• In-depth understanding of PPI interface at the single amino acid level
• Generation of invaluable tools for functional studies
• Exploit results to conduct an ULTImate Y2H™ screen or 1by1 Y2H assays
  
  

Our 1by1 interaction service features: 1) validating the interaction between two proteins identified in a Y2H screen or by other approaches, 2) mapping the interacting domain of a protein and 3) comparing the binding of wild-type and mutant proteins or between isoforms.

Molecules Tested:

• Wild-type and mutant full-length proteins
• Protein fragments and peptides
• Cytoplasmic and extracellular proteins
• Loops, tails and membrane-associated proteins

Key-benefits

• Simple, fast and robust pairwise interaction assays
• In-depth characterization of the PPI and domains involved
• Publication-grade illustrations including all positive and negative controls

We provide our customers with Next Generation Sequencing (NGS) analysis to accommodate their needs.

While traditional Sanger sequencing is the most reliable and qualitative tool for the majority of our Y2H projects there are exceptions.
Some baits naturally interact with several highly abundant targets in a Y2H screen, which can obscure the detection of low affinity and/or rare protein binding partners. The selection pressure is increased to reduce the number of positive clones that can be analyzed by standard sequencing, which can result in the loss of low affinity and rare interactions. In such a case, we propose NGS in order to uncover all potential interactors.

Key Benefits

• Generation of high-quality NGS dataset; no articifial amplication of background
• Low affinity and low abundant interactions identified

Our deliverables are presented and organized in a user-friendly format.

The results of each ULTImate screen contain three complementary files:

A Results summary with the identified protein partners, the detailed fragments and our confidence score- the Predicted Biological Score, PBS®.

An Excel® table with the sequences and the Genbank accession number and relevant IDs from e.g. Flybase, Wormbase, TAIR.

The ‘DomSight’ contains a graphical comparison of the experimental interacting domain for each partner and the Smallest Interacting Domain SID®, with known functional and structural domains.