A simple and rapid method of sample preparation from culture filtrate of M. tuberculosis for two-dimensional gel electrophoresis

Kumar, Gyanendra; Shankar, Hari; Bisht, Deepa; Sharma, Prashant; Singhal, Neelja; Katoch, Vishwa Mohan; Joshi, Beenu

doi:10.1590/s1517-83822010000200005

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References 19 publications

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“…Culture filtrate proteins were prepared as described earlier. 13,14 In brief, culture was centrifuged at 18,000 × g for 30 min at 4 °C and sequentially filtered through a 0.45-µm followed by a 0.22-µm Millex GV PVDF membrane (Millipore, Bedford, MA). Then, 10% sodium dodecyl sulfate (SDS) (Sigma, St. Louis, MO) was added to obtain 0.1% final concentration (w/v) in culture filtrate (CF) and kept in a boiling water bath for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Proteomics of Culture Filtrate of Prevalent Mycobacterium tuberculosis Strains: 2D-PAGE Map and MALDI-TOF/MS Analysis

et al. 2017

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Although diverse efforts have been done to identify biomarkers for control of tuberculosis using laboratory strain Mycobacterium tuberculosis H37Rv, the disease still poses a threat to mankind. There are many emerging M. tuberculosis strains, and proteomic profiling of these strains might be important to find out potential targets for diagnosis and/or prevention of tuberculosis. We evaluated the comparative proteomic profiling of culture filtrate (CF) proteins from prevalent M. tuberculosis strains (Central Asian or Delhi type; CAS1_Del, East African-Indian; EAI-3 and Beijing family) by 2D polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. As a result, we could identify 12 CF proteins (Rv0066c, Rv1310, Rv3375, Rv1415, Rv0567, Rv1886c, Rv3803c, Rv3804c, Rv2031c, Rv1038c, Rv2809, and Rv1911c), which were consistently increased in all prevalent M. tuberculosis strains, and interestingly, two CF proteins (Rv2809, Rv1911c) were identified with unknown functions. Consistent increased intensity of these proteins suggests their critical role for survival of prevalent M. tuberculosis isolates, and some of these proteins may also have potential as diagnostic and vaccine candidates for tuberculosis, which needs to be further explored by immunological analysis.

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Section: Methodsmentioning

confidence: 99%