Affinity Chromatography Lab Quiz

What is being isolated?

Isolate Lactate Dehydrogenase (using affinity chromatography)

Column Type

Ni-NTA column; Gel-like sepharose matrix with Ni ions linked, and held in place by NTA, which is covalently bound to sepharose 

What comes after LDH isolation?

SDS-PAGE to determine molecular weight of Histidine-Tagged-LDH

Affinity Chromatography

Purifies proteins using specific protein-ligand interactions

Affinity Chromatography Interactions with LDH

6x Histidines tagged on LDH (poly-histidine fusion protein) interact with metal ion ligands

How are his-containing proteins eluted? 

Intermediate imidazole causes weakly-bound non-specific His containing proteins to elute; High imidazole causes elution of his-tagged LDH (target protein) 

Affinity Chromotography Stationary and Mobile Phase

Stationary phase is resin with bound ligand; Mobile phase is buffer with competitive ligand (imidazole)

What sticks to resin in affinity chromatography?

Proteins with specific tag or strong binding affinity to the column

Western Blot Setup 

Membrane with target protein, Primary antibody, Enzyme-conjugated secondary antibody, enzyme substrate, colometric detection signal 

Lactate Dehydrogenase

Catalyzes a reversible, redox reaction to convert pyruvate to lactate

LDH Structure

Quaternary; Four subunits (tetramer) composed of two subunits M (in anaerobic conditions) and H (in aerobic conditions)

5 Possible Isozymes of LDH

M4, M3H, M2H2, MH3, H4