Henderson-Hasselbach Equation - ANSWER pH = pKa + log ([A-] / [HA]) FMOC Chemical Synthesis - ANSWER Used in synthesis of a growing amino acid chainto a polystyrene bead. FMOC is used as a protecting group on the N-terminus. Salting Out (Purification) - ANSWER Changes soluble protein to solid precipitate. Protein precipitates when the charges on the protein match the charges in the solution. Size-Exclusion Chromatography - ANSWER Separates sample based on size with smaller molecules eluting later. Ion-Exchange Chromatography - ANSWER Separates sample based on charge. CM attracts +, DEAE attracts -. May have repulsion effect on like charges. Salt or acid used to remove stuck proteins. Hydrophobic/Reverse Phase Chromatography - ANSWER Beads are coated with a carbon chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent (acetonitrile). Affinity Chromatography - ANSWER Attach a ligand that binds a protein to a bead. Elutewith harsh chemicals or similar ligand. SDS-PAGE - ANSWER Uses SDS. Gel is made from cross-linked polyacrylamide. Separates based off of mass with smaller molecules moving faster. Visualized with Coomassie blue. SDS - ANSWER Sodium dodecyl sulfate. Unfolds proteins and gives them uniform negative charge. Isoelectric Focusing - ANSWER Variation of gel electrophoresis where protein chargematters. Involves electrodes and pH gradient. Protein stops at their pI when neutral. FDNB (1-fluoro-2,3-dinitrobenzene) - ANSWER FDNB reacts with the N-terminus of the protein to produce a 2,4- dinitrophenol derivative that labels the first residue. Can repeat hydrolysis to determine sequential amino acids. DTT (dithiothreitol) - ANSWER Reduces disulfide bonds. Iodoacetate - ANSWER Adds carboxymethyl group on free -SH groups. Blocks disulfide bonding