Protein Purification Flashcards
What is Ion Exchange chromatography?
Separates proteins by charge - all proteins have a net charge at any given pH. Column is packed with charged resin –> is washed to collect flow through with buffer –> pH is changed or salt is added to elute the protein. Anion or cation exchange based on what the protein binds to.
What is gel filtration (size exclusion) chromatography?
Separates molecules by size and shape. Column is packed with cross-linked gel (porous beads). Protein mixture is added to column –> Wash and elution (proteins move through column, DO NOT BIND, and seperate according to size) (large proteins move through column quickly and elute first)
What is hydrophobic interaction chromatography?
Proteins contain hydrophobic amino acids that may be exposed at the surface of the protein and interact with other hydrophobic molecules. The beads will covalently attach to hydrophobic molecules. The proteins will be elutes by reducing ionic strength by adding a buffer WITHOUT salt.
What is affinity chromatography?
Separate by specify. Column is packed with molecules (ligands) that interact strongly with protein of interest. The most specific form of chromatography. Protein mixture is added to column –> Wash and molecules of intrest bind to column, and other proteins flow through with buffer –> Elute the proteins by adding high concentration of ligand –> use dialisis to get protein from ligand.
What is centrifugation?
Separates large from small particles (or based on density). Separate sub cellular fractions, isolate specific organelles, or isolate all soluble proteins in a cell after lysis. Once the protein-containing solution (supernatant) is separated from the rest of the cellular debris, the protein of interest can be purified.
What is gel electrophoresis?
Separation of proteins, nucleic acids, etc., by size, shape, and charge. Proteins migrate based on their charge to mass ratio. Proteins visualized by radioactivity or staining. Use gels made of crosslinked polymer (polyacrylamide) or solidified agarose. (Anode (-) at top and Cathode (+) at bottom).
What is SDS-PAGE ?
Denature proteins by adding SDS (-) makes proteins migration rate depend solely on molecular weight. SDS eliminates charge and shape differentiation and site on the denatured protein and creates a negative charge. Will tell you how big and pure a protein is.
What is Native PAGE?
Polyacrylamide Gel Electrophoresis is finding the protein’s shape based on its native state. Most common reducing agents used are DTT and betaME.
