IV. Determination Of Protein Linkage Yeast Two-Hybrid System Co-Immunoprecipitation

4.1. Yeast Two-Hybrid System Interactome is the whole set of molecular interactions in cells. When spoken in terms of proteomics, it refers to the whole set of protein-protein interactions in cells.

Signals (Development, Temperature, Drought, & High Salts) ABA Receptor? abi1 (PP2C) Kinase Cascade? abi2 (PP2C) AtDBKs abi3 AtDPBFs/ABFs abi4 abi5 lea Gene Dc3

The Properties of The Two-Hyb Clones Screened by AtDPBFl (abi5) Clone Number 1 6 42 65 Size 285 aa 121 aa 201 aa 191 aa Gene Name DBK1 DBK2 Gene Size (Full ORF) 319 aa 293 aa BAC Clone TIE8, T24B2, F14P3 T1B9, T8D2, T8C13, T13C19 Intron No 1 (156bp) Localization Chr 3 between 13600-14100kb Interact with AtDPBF1 Yes Interact with AtDPBF2 Interact with AtDPBF3 Interact with AtDPBF4 Interact with GBF4 Interact with AtPP2CA Activate AtDPBF1 without Gal4-AD Activate AtDPBF3 without Gal4-AD Activate At4D2 without Ga14-AD Activate At4D7 without Gal4-AD Bound Sequence TLED/TLGE RQPT Activate AtDPBFI if T201to S201 Binding Activitv if T201to S201 Activate AtDPBFl if T206 to A206 Binding Activity if T206 to A206 Activate AtDPBFl if T47 to A47 & T206 to A206 Binding Activitv if T47 to A47 & T206 to A206 Activate AtDPBFl if T201 to S201 & T206 to A206 Binding Activity if T201 to S201 & T206 to A206 Activate AtDPBFl if T47 to A47, T201 to S201 & T206 to A206 Binding Activity if T47 to A47, T201 to S201 & T206 to A206 Activity is repressed by AtPP2CA

4.2. Co-Immunoprecipitation Coprecipitation of proteins from whole-cell extracts is a valuable approach to test for physical interactions between proteins of interest. When a precipitating antibody is used, this method is referred to as co-immunoprecipitation. Coprecipitation can be used to study interactions between known proteins under a variety of conditions and as a means of identifying components of a complex.

In a typical experiment, cells are lysed and a whole-cell extract is prepared under nondenaturing conditions. The protein is precipitated from the lysate with a solid-phase affinity matrix and the precipitate is tested for the presence of a second specifically associated protein. The approach can be used for native or epitope-tagged proteins for which antibodies are available, or for recombinant proteins that have been engineered to bind with high affinity to a molecule that can be coupled to a solid-phase matrix. Two frequently used epitopes are derived from influenza hemagglutinin protein (HA) and human c-Myc and are recognized by high affinity mAbs. The presence of an associated protein is detected by separating the precipitated proteins by SDS-PAGE and immunoblotting with a second antibody that recognizes the putative associated protein. Controls to test specificity of interaction are crucial.

Prepare duplicate samples in microcentrifuge tubes on ice: 0.5 to 1mg whole-cell extract 1μg antibody 5M NaCl to equalize at 100mM NaCl Co-immunoprecipitation buffer to 0.5 ml final volume. Invert tube gently several times and incubate on ice for 90 min with occasional tube inversion. Microcentrifuge 10 min at maximum speed, 4℃, to pellet nonspecific aggregates. Transfer supernatant to a new microcentrifuge tube. Add 50 μl of protein A– or protein G–Sepharose slurry (25 to 30 μl bead volume). Rotate tube gently at 4 ℃ for 30 to 60 min. Gently pellet protein A/G–Sepharose by centrifuging 30 sec at 1000 rpm in a tabletop centrifuge, 4 ℃. Wash pellet three times with 1 ml co-immunoprecipitation buffer. Aspirate as much liquid as possible from the final without touching the beads and add 25μl of 2× sample buffer. Prepare for SDS-PAGE analysis by boiling for 5 min, vortexing, and microcentrifuging briefly to pellet beads. Load eluates onto an SDS-polyacrylamide gel, arranging duplicate samples to allow preparation of duplicate blots. Separate by electrophoresis. Immunoblot duplicate samples separately with antibodies for each of the two proteins. Be sure to include aliquots of the whole-cell extract for comparison and as a positive control for the immunoblot. Protein A is a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in biochemical research because of its ability to bind immunoglobulins. It binds proteins from many of mammalian species, most notably IgG’s. It binds with the Fc region of immunoglobulins through interaction with the heavy chain. The result of this type of interaction is that, in serum, the bacteria will bind IgG molecules in the wrong orientation (in relation to normal antibody function) on their surface which disrupts opsonization and phagocytosis. Protein G is an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria much like Protein A but with differing specificities. It is a 65-kDa (G148 protein G) and a 58 kDa (C40 protein G)[1] cell surface protein that has found application in purifying antibodies through its binding to the Fc region. The native molecule also binds albumin, however, because serum albumin is a major contaminant of antibody sources, the albumin binding site has been removed from recombinant forms of Protein G. Protein L is a 36,000 dalton immunoglobulin-binding protein isolated from the bacteria Peptostreptococcus magnus. Unlike Protein A and Protein G, which bind to the Fc region of immunoglobilins (antibodies), Protein L binds antibodies through light chain interactions. Since no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of antibody classes than Protein A or G. Protein L binds to representatives of all antibody classes, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (ScFv) and Fab fragments also bind to Protein L.