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FIGURE 1. Towards the generation of a proteome-scale human yeast two-hybrid map.

From the following article:

Towards a proteome-scale map of the human protein–protein interaction network

Jean-François Rual, Kavitha Venkatesan, Tong Hao, Tomoko Hirozane-Kishikawa, Amélie Dricot, Ning Li, Gabriel F. Berriz, Francis D. Gibbons, Matija Dreze, Nono Ayivi-Guedehoussou, Niels Klitgord, Christophe Simon, Mike Boxem, Stuart Milstein, Jennifer Rosenberg, Debra S. Goldberg, Lan V. Zhang, Sharyl L. Wong, Giovanni Franklin, Siming Li, Joanna S. Albala, Janghoo Lim, Carlene Fraughton, Estelle Llamosas, Sebiha Cevik, Camille Bex, Philippe Lamesch, Robert S. Sikorski, Jean Vandenhaute, Huda Y. Zoghbi, Alex Smolyar, Stephanie Bosak, Reynaldo Sequerra, Lynn Doucette-Stamm, Michael E. Cusick, David E. Hill, Frederick P. Roth and Marc Vidal

Nature 437, 1173-1178 (20 October 2005)

doi: 10.1038/nature04209

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a, Schema of the high-throughput yeast two-hybrid pipeline. Individual steps (middle column) and representative examples (flanking left and right columns) are indicated. The top panel of the left column represents the matrix of all protein pairs. All available ORFs from human ORFeome v1.1 were transferred into both DB and AD vectors by recombinational cloning (middle panel of left column). The top panel of the right column shows the mating process, with each bait mated to individual pools of 188 AD-ORFs. Initial phenotypic testing evaluated growth of diploid cells on selective medium in response to enhanced levels of the GAL1::HIS3 selective marker (bottom panel of left column). All positive diploids from phenotyping no. 1 (red circles) were subsequently tested for activation of both GAL1::HIS3 and GAL1::lacZ reporter genes. Auto-activators were identified by growth on medium containing cycloheximide (bottom panels of left and right columns). Positive colonies from phenotyping no. 2 (outlined in red) were isolated and used to PCR-amplify both DB-ORF and AD-ORF fragments for sequencing. b, Verification of yeast two-hybrid interactions by co-affinity purification assays. Fifteen representative examples of co-affinity purification-positive assays are shown. The middle and bottom panels show expression controls of Myc–prey and GST–bait fusion proteins, respectively. Each lane pair in the top panels shows presence or absence of Myc–prey fusions after affinity purification, demonstrating binding to GST–bait fusion proteins (+ ) or to GST alone (- ). The Table summarizes the data obtained for four different classes of protein pairs. 'Y2H and LCI' describes interactions reported in both the yeast two-hybrid and LCI data sets. 'Y2H/LCI-negative' describes pairs of proteins that were not reported to interact either in the yeast two-hybrid or in the LCI data sets. Rows indicate the total number of interactions tested and considered for scoring (Total), the number of interactions not verified by co-affinity purification (co-AP-), the number of interactions verified by co-affinity purification (co-AP+), the proportion of co-affinity purification-positive interactions (success rate), and the adjusted success rate (which accounts for the observation that one-third of all co-affinity purification experiments yield an apparently positive result without regard to whether or not the protein pair truly interacts; see Supplementary Data IX). Identities, lane positions and scoring of all protein pairs tested by co-affinity purification are provided in Supplementary Tables S2 and S3.

Figures & Tables index
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