Purification of Bacteriophages and SDS-PAGE Analysis of Phage Structural Proteins from Ghost Particles
互联网
479
Concentration and purification of infectious particles are prerequisites for structural and functional characterization of bacteriophages. The methods detailed in the first part of this chapter outline the protocols commonly used to obtain purified phages: the concentration of phage particles by precipitation with polyethylene glycol and their purification by centrifugation in CsCl step gradients and subsequently by equilibrium centrifugation. This sequence of procedures, if carried out as a whole, ensures a purification of high quality, which is well suited for most analytical techniques used to characterize bacteriophage particles.
The second part of this chapter describes the preparation of “ghosts” or DNA-less bacteriophages. These particles should be preferred to the entire bacteriophages for one-dimensional SDS-PAGE analysis of phage structural proteins, since running of the phage proteins through the gel is not disturbed by the presence of the phage DNA. This allows an optimal resolution, which is necessary for proteomic approaches such as -terminal protein sequencing or mass spectrometry using proteins isolated from distinct gel bands.
