Affinity Purification of Interacting Proteins from Cell Lysates Protocol Recombinant protein or a chemically synthesized bioactive fragment is immobilized on resin and used as a probe to capture interacting proteins directly from a cell extract. Affinity-purified proteins are fractionated by gel electrophoresis and visualized by Coomassie staining. Proteins that interact specifically are identified by comparing this gel profile to one obtained from cell lysates passed over a control resin lacking the immobilized probe protein.
Lectin-Agarose Affinity Chromatography Protocol Two methods are provided for purifying glycoproteins using wheat-germ agglutinin or concanavalin A-Sepharose. Because lectin-affinity matrices can bind a few milligrams of protein per milliliter of affinity matrix, only a small amount of affinity gel matrix is required. The batchwise method is recommended when protein volume is large.
Overview of Affinity Purification in Combination with Mass Spectrometry Protocol Protein complexes can be isolated by several different approaches. For example, a protein can be tagged with an epitope such as Flag or TAP and then overexpressed in a target cell, allowing the interacting proteins to be purified. Similarly, epitope tags can be homologously recombined into the endogenous locus ("knocked-in"), allowing protein complexes containing the tagged proteins to be isolated at their natural expression level.
Protein Ligand Affinity Chromatography Protocol Protocol describes the coupling of peptides or proteins to Affi-Gel 10 (Bio-Rad). Affi-Gel 10 is a cross-linked agarose derivatized with N-hydroxysuccinimide (NHS) ester on a 10-atom-long spacer arm. The NHS ester allows spontaneous coupling of proteins via free amino groups.
Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography Protocol Recombinant proteins engineered to have a polyhistidine tail at either the carboxyl or amino terminus can easily be purified in one step by affinity chromatography on a resin carrying chelated nickel ions. Chromatography can be carried out in column or batch formats. After unbound proteins are washed away, the target protein is eluted using imidazole, which typically preserves the antigenic and functional features of the protein.