BIOC23H3 Lecture : Lec 8 - Affinity Chromatography.doc
2 May 2012
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Document Summary
Specialized adsorption chromatography; specific physical binding between 2 solutes. Antibody = to purify antigen = expensive and non-specific binding effects: matrix = large pore size. After activation, beads can react with ligand. Steric interference btn matrix and target moc. Therefore use spacer arm: usu hydrophilic, agarose matrix bead spacer arm small ligand bind interest protein. Load sample: column equilibrium, wash away unwanted proteins, elute interest protein, column regeneration. Elution strategies: specific elution = add biospecific analogue, ligand, antigen, antibody to elute interest protein; eluant still in active form, competitive strategies: Elution by displacement using free ligand: target protein binds to free ligand and the target protein is therefore displaced and elutes. Fusion protein tags: gst, 26 kda protein, will bind reduced glutathione, 2 tags, tap tag: Or can put antibody to matrix and antibody will bind all proteins that contain epitope tag. Lac operon: promoter = drives expression, operon = binding site of repressor.
