Experiment 7: Protein Purification, Chromatography and SDS-PAGE

Chromatography

process that are degsigned to separate different molecules

Stationary phase

immobile, resides on outer edge (usually), interact at varying degrees with molecules in mobile phase, inert matrix with functional groups attached

Mobile phase

moving, molecules in can be immobilized by stationary phase, filled with molecules that move through stationary phase, oftern buffer/protein mixtures

fractions

different collect samples as mobile phase exits stationary phase, consists of different components

Types of Protein Chromatography

Ion-exchange, affinity, size exclusion

Ion exchange chromatography (IEX)

based reversible electrostatic interations between charged molecules, proteins conatin different charges at different pH, bound protein eluted by changing pH or salt conditions

Affinity chromatography (AC)

based specific and reversible high affinity interaction with ligand, wash buffer (clear others out), elution buffer (elute protein)

Tags

sequence added to amino acid to bind affinity column, His-tag most common (6-8 his residues)

Affinity wash buffer

promote flow of proteins, prevent column from drying out

Affinity elution buffer

high [] of molecule that outcompetes ligand, elutes protein

Elution faction composition

buffer, protein of interest, ligand(much smaller) used to remove protein

Size exclusion chromatography (SEC)/gel filtration

seperates bases size of molecules, porous beads of varying size, larger elutes first (skips the beads) and smaller elutes last (travel through beads)

SEC for analytical technique

determine whether monomer/dimers/complex

native structure fof protein

inherent shape/size/charge of protein

SDS-PAGE

electrophoresis, separates protein based on polypeptide chain length, evulate protein purity, discontinous gel (stacking or resolving)

SDS-PAGE sample preparation

sample heated for 5 min at 95C in loading buffer, all proteins become negatively charged rod shapes of different lengths

loading buffer

Beta-mercaptoethanol and high tempeature denature protein, SDS uniformly cover protein with negative charges, sucrose or glycerol to sink to bottom of well

Polyacrylamide gels

formed by polymerization of acrylamide into polyacrylamide, coupled with crosslinkingbisacrylamide, catazlyed by TEMED (free radical), mesh framework

resolving gel (separating)

6-15% acrylamide, bottom portion

stacking gel

4% acrylamide, function to focus protein into tight band before separation

Visualizing the gel

stain and destain, most common Coomassie based, time consuming but high sensitivity

purity of protein

pure = 1 single band