Protein Methods

Unit 1 - BIOS 301 Rice

Preparation and Bulk Purification (2)

Salting out & Dialysis

Chromatographic Purification (separation based on physical properties) (5)

Charge/ HB: Anion/ Cation Exchange, RPC, HIC

Size and Binding: Gel Filtration (Size-Exclusion), Affinity

Quantification (how much do I have, not purity) (4)

Absorbance (A260/A280), Bradford, Biuret, Lowry (colorimetric)

Analytic (is this the only thing there)

SDS-PAGE, Native Gel Shift, IEF, 2D electrophoresis (complex protein mix)

Specific Detection and Identification

Western Blot, ELISA

Exact MW and postranslational modifications

Top down and bottom up mass spec

Bradford

Coomassie dye binds to basic and hydrophobic amino acids, shifting from a free to bound state (blue)

Biuret

at alkaline pHs, Cu2+ binds to the amide N, reducing Cu2+ to Cu, yielding a purple color that can be quantified

Lowry

uses Biuret and the FC reagent that reacts with aromatic amino acids prev. modified by Cu, intensifying blue color

SDS-PAGE (use Western Blot to check)

separation based on mass. denature proteins, and SDS coats the surface, imparting a uniform negative charge. ladders to identify MW (multiple bands indicate subunits)

Gel Shift Assay

native state to see if protein is still functional or binding to DNA (Kd can be determined)

Western Blot (after SDS-PAGE)

uses antibodies to find specific protein on a membrane after SDS-PAGE

ELISA

antibody detection

coat surface with antigens —> add nonspecific proteins to fill in gaps and eliminate false positives —> add primary antibody to bind to target antigen —> add secondary antibody (anti-antigen) —> add substrate that reacts with secondary antibody to produce color

darker color means more protein

Top-Down MS

liquid chromatography peptide separation of whole protein —> electrospray ionization —> ionized intact proteins —> MS —> proteoform selection and analysis —> bioinformatic database search

Bottom-Up MS

digest proteins (usually trypsin) —> liquid chromatography peptide separation of digested peptides —> electrospray ionization —> ionized intact proteins —> MS —> peptide selection and analysis —> bioinformatic database search