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The main interest of the double screening by Phage Display and Yeast Two-Hybrid is to enrich the selected antibodies in what we call Intrabodies, i.e An Intrabody is an antibody functional inside the cell.
Intrabodies are fantastic tools for live cell imaging like video-microscopy and single molecule localization or in vivo applications. Learn more about Hybrigenics' Intrabody selection service.
Pre-selected hs2dAb are transferred from phages to a Y2H prey vector by Gap Repair. This vector allows the fusion of the Activation Domain (AD) of a transcription factor to the single-domain antibody sequence. This Y2H Antibody library is then transformed into a genetically modified yeast strain lacking the HIS3 gene necessary for the synthesis of histidine. The antigen of interest is fused to the DNA Binding Domain (DBD) of a transcription factor in a Y2H bait vector and then transformed into a second yeast strain of opposite sexual type compared to the prey yeast strain.
Put together, the two yeast strains mate and form diploids expressing both the Antigen and a VHH antibody from the library. If the antibody recognizes the antigen, it allows for the reconstitution of the functional transcription factor. It activates the transcription of the HIS3 reporter gene and allows yeast cells to grow on a selective medium lacking histidine. The DNA of the positive clones is then sequenced and analyzed to identify the selected intrabodies.
Hybrigenics has an unparalleled experience of more than 8,000 Y2H screens and improved this technology to its highest level. Optimizations include technical improvements like yeast strains engineering and patented mating protocol. Our state-of-the-art Y2H Yeast Two-Hybrid technology allows for the selection of the functional intrabody you need for in cell applications.
Independently from hs2dAb library screening for intrabody selection, Hybrigenics builds its own cDNA libraries and offer to screen them for the identification of binding partners of your favorite protein, DNA, RNA or Small Molecule. We have almost 100 libraries from 25 different species and can build specific ones for your project upon request. They are the most complex and diverse Y2H libraries available on the market.