Comparative purification of a Salmonella bacteriophage for protein profiling.
- 1Research Institute for Biological Safety Problems, Gvardeyskiy, Kazakhstan.
- 2Ulyanovsk State Agrarian University Named After P. A. Stolypin, Ulyanovsk, Russia.
- 3Research Institute for Biological Safety Problems, Gvardeyskiy, Kazakhstan. [email protected].
Abstract
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.
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BibTeX
@article{toleukhan2026comparative,
title = {{Comparative purification of a Salmonella bacteriophage for protein profiling.}},
author = {Alinur Toleukhan and Akbope Abdykalyk and Aktoty Anarbekova and Azamat Abdimukhtar and Bekzat Yerzhigit and Nazym Syrym and Nadezhda Zinina and Sabira Alpysbayeva and Makhpal Sarmykova and Yeldos Serikbay and Kali Tileukhanov and Nurlan Kozhabergenov and Bekbolat Usserbayev and Yeraly Shayakhmetov and Kuandyk Zhugunissov and Sergazy Nurabayev and Andrey Shestakov and Bolat Yespembetov},
journal = {Applied microbiology and biotechnology},
year = {2026},
month = jul,
doi = {10.1007/s00253-026-13982-z},
url = {https://europepmc.org/article/MED/42909619},
note = {CC BY},
}
RIS
TY - JOUR
TI - Comparative purification of a Salmonella bacteriophage for protein profiling.
AU - Alinur Toleukhan
AU - Akbope Abdykalyk
AU - Aktoty Anarbekova
AU - Azamat Abdimukhtar
AU - Bekzat Yerzhigit
AU - Nazym Syrym
AU - Nadezhda Zinina
AU - Sabira Alpysbayeva
AU - Makhpal Sarmykova
AU - Yeldos Serikbay
AU - Kali Tileukhanov
AU - Nurlan Kozhabergenov
AU - Bekbolat Usserbayev
AU - Yeraly Shayakhmetov
AU - Kuandyk Zhugunissov
AU - Sergazy Nurabayev
AU - Andrey Shestakov
AU - Bolat Yespembetov
PY - 2026
DA - 2026/07/01
JO - Applied microbiology and biotechnology
DO - 10.1007/s00253-026-13982-z
UR - https://europepmc.org/article/MED/42909619
AB - 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.
SN - 0175-7598
SN - 1432-0614
ER -