LSCI 211 Lecture Notes - Lecture 8: Affinity Chromatography, Column Chromatography, Cross-Link
28 Aug 2016
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Specific activity = total activity / amount (mg) of a protein. Adding soluble salts disrupts the spheres of water around protein molecules. Size/charge/affinity- gel permeation / ion exchange / ligand or antibody chromatography. Stationary phase- a support matrix with components to interact. Mobile phase- a solvent that promotes differential partitioning. Plate is coated with capture antibody, antigen binds, detecting antibody binds, antibody with enzyme binds, substrate is added and converted to detectable form. Different types of chromatography can be used individually or in succession. Protein mixture is passed through a cross-linked polymer. Larger molecules pass through more quickly, bypassing the polymer. Cation exchangers- more negatively charged proteins move more slowly. Anion exchanges- more positively charged proteins move more slowly. Separates molecules by affinity through a specific polymer-bound ligand. Unwanted proteins are washed through the column, desired protein sticks. Ligand solution is then passed through to detach (elute) the desired protein.
