Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin. Coevolution between nonhomologous but functionally similar proteins and their conserved partners in the Legionella pathogenesis system. FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA binding protein. BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B. Wang, B., Pelletier, J., Massaad, M.J., Herscovics, A. Fu, H.) (Humana Press, Totowa, New Jersey, 2004). In Protein–Protein Interactions: Methods and Applications. Glutathione- S-transferase-fusion based assays for studying protein-protein interactions. Studying protein-protein interactions via blot overlay or far western blot. A quantitative protein interaction network for the ErbB receptors using protein microarrays. Surface plasmon resonance imaging as a tool to monitor biomolecular interactions in an array based format. A generic protein purification method for protein complex characterization and proteome exploration. Gene function prediction from congruent synthetic lethal interactions in yeast. Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Gordon, G.W., Berry, G., Liang, X.H., Levine, B. A novel genetic system to detect protein-protein interactions. A contingent replication assay for the detection of protein-protein interactions in animal cells. Vasavada, H.A., Ganguly, S., Germino, F.J., Wang, Z.X. A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).ĭmitrova, M. In Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2005). Protein–Protein Interactions: A Molecular Cloning Manual 2nd edn. (Humana Press, Totowa, New Jersey, 2004). Protein-Protein Interactions: Method and Application. Typically, 2–3 d are required to carry out the experiment.įu, H. Furthermore, in cases where they bind to each other indirectly, Far WB allows the examination of candidate protein(s) that form a complex between them. Unlike most methods using cell lysates (e.g., co-immunoprecipitation (co-IP)) or living cells (e.g., fluorescent resonance energy transfer (FRET)), Far WB determines whether two proteins bind to each other directly. Compared with other biochemical binding assays, Far WB allows prey proteins to be endogenously expressed without purification. The bait proteins are detected on spots in the membrane where a prey protein is located if the bait proteins and the prey protein together form a complex. The membrane is then blocked and probed, usually with purified bait protein(s). The proteins in the membrane are then denatured and renatured. In Far WB, proteins in a cell lysate containing prey proteins are firstly separated by SDS or native PAGE, and transferred to a membrane, as in a standard WB. Far western blotting (WB) was derived from the standard WB method to detect protein–protein interactions in vitro.
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