Classic Protocol in 2004

Glutathione-S-transferase (GST) fusion proteins have had a range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria¹. Typically, GST pull-down experiments are used to identify interactions between a probe protein and unknown targets and to confirm suspected interactions between a probe protein and a known protein^2,3. The probe protein is a GST fusion, whose coding sequence is cloned into an isopropyl-β-D-thiogalactoside (IPTG)-inducible expression vector. This fusion protein is expressed in bacteria and purified by affinity chromatography on glutathione-agarose beads. Target proteins are usually lysates of cells, either labeled with [³⁵S]methionine or unlabeled, depending on the method used to assay the interaction between the target and the probe. The cell lysate and the GST fusion protein are incubated together with glutathione-agarose beads. Complexes recovered from the beads are resolved by SDS-PAGE and analyzed by western blotting, autoradiography or staining.

The 'megaprimer' method of site-directed mutagenesis uses three oligonucleotide primers and two rounds of polymerase chain reaction (PCR)¹. One oligonucleotides is mutagenic; the others are forward and reverse primers that lie upstream and downstream from the binding site for the mutagenic oligonucleotide. The mutagenic primer and the nearer of the external primers are used in the first PCR to generate and amplify a mutated fragment of DNA. This amplified fragment—the megaprimer—is used in the second PCR in conjunction with the remaining external primer to amplify a longer region of the template DNA. This protocol is based on a method that uses forward and reverse external primers with significantly different melting temperatures (T_m)².

Southern transfer and hybridization¹ is used to study how genes are organized within genomes by mapping restriction sites in and around segments of genomic DNA. This protocol describes the first stages of Southern blotting: digestion of genomic DNA with restriction enzymes, separation of the resulting fragments by gel electrophoresis, and capillary transfer of the denatured fragments to a membrane².