BIOS 300 Protein Purification/Characterization Methods

crude purification

type of purification based on physical or chemical properties (umbrella term)

chromatographic purification

type of purification based on specific interactions (umbrella term)

precipation

type of crude purification that utilizes varying salt concentrations to elute proteins by varying solubility

heat treatment

type of crude purification that uses heat to denature and precipitate contaminating proteins to isolate target protein

pH

type of crude purification that uses isoelectric point to elute protein out of solution

ion exchange

type of chromatographic purification that uses cation/anion matrix to attract a charged protein

affinity method

type of chromatographic purification that uses ligand that binds specifically and reversibly to target protein

gel filtration

type of chromatographic purification that works based on varying protein size (smaller proteins will take longer to elute)

hydrophobic

type of chromatographic purification that increases sodium concentration, forcing hydrophobic regions to bind, and then lowers Na concentrations so HP forces are weakened but they elute the solution first

fluorescence microscopy

type of characterization method where target protein is tagged with fluorophore and examined under fluorescence microscope to show protein location

molecular sieve

type of characterization method where proteins are characterized based on size or shape

mass spectrometry

type of characterization method where proteins are characterized by molecular size/sequence

biochemical

type of characterization method where proteins are characterized by function and assays that target function of TP

X-ray crystallography

provides 3D structure of crystallized structure *large molecules*

SDS-PAGE

type of characterization method where proteins are characterized based on molecular weight (proteins are unfolded and smaller proteins move through gel quicker)

Western Blotting

type of characterization method where proteins are separated through PAGE and then transferred to member which is blocked by generic protein and exposed to antibody

immunoprecipation

type of characterization method where a specific antibody isolates and enriches TP from mixture and TP is then eluted

NMR (nuclear magnetic resonance spectroscopy)

provides family of similar 3D structure *small molecules*