Purification of DNA-binding proteins using biotin/streptavidin affinity systems.
This unit presents purification protocols that exploit the tight and essentially irreversible complex that biotin forms with streptavidin. A DNA fragment containing a high-affinity binding site for the protein of interest is prepared and a molecule of biotinylated nucleotide is incorporated into one of the ends of the DNA fragment. The protein of interest is allowed to bind to the high-affinity recognition site present in the biotinylated fragment. The tetrameric protein streptavidin is then bound to the biotinylated end of the DNA fragment. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently removed by adsorption onto a biotin-containing resin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin-containing resin. Proteins remaining in the supernatant are washed away under conditions that maximize the stability of the DNA-protein complex. Finally, the protein of interest is eluted from the resin with a high-salt buffer. Both batch and column formats are presented, as is a protocol for the use of streptavidin-agarose. A support protocol describes a mobility shift assay for detecting sequence-specific DNA-binding proteins.
Chodosh
L A
LA
University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Buratowski
S
S
D001426:Bacterial Proteins / Q000737:chemistry*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D024202:Electrophoretic Mobility Shift Assay; D011485:Protein Binding
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066114:biotin-streptavidin complex; D001710:Biotin; D004247:DNA