Intro to Protein Column Chromatogrpahy

chromatography

processes that separate different molecules from each other for purposes of identification, isolation, and analysis

• made up of immobile stationary phase and a moving mobile phase

stationary phase

an inert matrix to which functional groups are covalently attached; usually beads made up of a polymer

mobile phase

buffer/protein mixture in protein column chromatography

ion exchange chromatography

based upon reversible electrostatic interactions between charged molecules and charged functional groups bound to inert resin beads

• for proteins w. different amino acid compositions

affinity chromtography

based upon the affinity between a protein and a ligand molecule that is linked to an insoluble support

key of specificity for affinity chromatography

finding a ligand that binds to your protein, but no other in the solution

His-tags

proteins with ___ will bind to columns that have a nickel ion immobilized on the column

wash buffer

used in affinity chromatography to promote the flow of protein and to prevent the column from becoming dry

size exclution chromatography

method that separates molecules on the basis of thier size

• inert beads

large small

in size-exclusion chromatography, ___ molecules move faster than ___

SDS-PAGE

electrophoresis method that separates proteins from each other on the basis of the length of thier polypeptide chains

rzns why SDS-PAGE separates proteins

1. all proteins are given the same q/f ratio

   1. gels are sieved though porous structural support

sample preparation for SDS-PAGE

sample heated to 95ºC in loading buffer; destroys 3D structure of protein

• SDS associates with each protein which makes then -vely charged

TEMED

catalyst of the polymerization of polyacrylamide

• causes the formation of free radicals in ammonium persulfate

stacking gel

4% acrylamide gel whose job it is to focus protein into a tight band before protein goes through resolving gel

Coomassie-blue stain

used to dye both the protein and the acrylamide

glacial acetic acid / methanol

used to destain acrylamide

lactate dehydrogenase

catalyzes the reversible rxn between pyruvate (NADH) (340 nm) and lactate (NAD+)