ST 2 - C. Discovering Phosphorylation Sites 1. radioactive...

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A. Characterizing Receptors 1. Receptor Gene Cloning: ligand will bind if receptor is expressed, cell will respond if required signaling pathways are activated 2. Affinity Chromatography: ligands are crosslinked to beads. Only receptors with high affinities are retained, and then eluted after washing 3. Fluorescence Activated Cell Sorting (FACS): light scattering measures cell size and viability, light emission for fluorescent labeled antibody measures receptor expression. Cells sorted in single charged droplets 4. Flourescently labeled antibody – recognizes only a specific receptor. II. Components of Signalling Pathways A. Phosphatase: replace phosphates with side chain hydroxyl. B. Kinase: opposite of phosphatase
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Unformatted text preview: C. Discovering Phosphorylation Sites 1. radioactive inorganic phorphorous isotopes 2. mass spec 3. kinase assay 4. immunobloting with antibodies to the phosphorylated site D. GTP: controls G protein activation 1. glycine and threonine bind phosphate of gamma P of GTP = conformational change 2. GTPases hydrolyzes GTP = relaxed state E. Trimeric G-Proteins 1. alpha and gamma tethered to mem covalently by lipids 2. beta gamma regulatory 3. alpha binds GDP/GTP F. Lipid Derivatives: Secondary Messengers 1. PIP2 -> DAG + IP3 2. DAG: activates PKC, remains in cystolic leaflet of membrane 3. IP3 is released into cytosol, opens Ca2+ channels in ER, Ca synergizes with DAG...
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