Comparative purification of a Salmonella bacteriophage for protein profiling

Salmonella enterica bacteriophages are increasingly studied for bacterial biocontrol and applied microbiology. However, reliable downstream characterization depends strongly on how the phage material is purified and concentrated. This study compared five preparation strategies for a Salmonella bacteriophage: sucrose density gradient centrifugation, cesium chloride gradient centrifugation, ultrafiltration followed by lyophilization, ultrafiltration followed by ammonium sulfate precipitation, and ultrafiltration followed by trichloroacetic acid precipitation. The preparations were assessed for infectious phage recovery, total protein concentration, SDS-PAGE protein profiles, an exploratory molecular weight-based comparison with a reference phage dataset, and targeted real-time PCR screening for selected virulence-associated determinants. Ultrafiltration followed by ammonium sulfate precipitation gave the highest infectious phage recovery among the infectivity-preserving workflows and produced a consistent eight-band SDS-PAGE profile. Trichloroacetic acid precipitation produced a similar banding pattern but was considered mainly a protein precipitation method because of its denaturing effect. Cesium chloride gradient centrifugation gave the most complex SDS-PAGE profile, while ultrafiltration followed by lyophilization gave the least resolved one. Several bands fell within the molecular-mass ranges reported for proteins of related Salmonella phages, but matching by molecular mass alone cannot identify the proteins, which would require LC-MS/MS. Targeted real-time PCR did not detect sopE, sspH1, sseI, or sopE2. Under the conditions tested, ultrafiltration followed by ammonium sulfate precipitation gave the highest infectious recovery and a consistent SDS-PAGE pattern; these results describe how the workflows behaved here rather than showing that one method is generally superior. KEY POINTS: • Five workflows were compared for Salmonella phage purification. • Ammonium sulfate precipitation gave the highest infectious recovery. • Selected virulence-associated genes were not detected by qPCR.