BCG strain overexpressing the toxin (vapC12)–antitoxin (vapB12)-His(6X) locus was used for sample preparation. Log phase culture of the overexpressed strain was washed with PBS and inoculated in minimal media with 0.1% glycerol and minimal media with 0.01% cholesterol. The cultures were allowed to grow for 48 hrs and cell lysate was prepared. Immunoprecipitation was performed using an anti-His antibody (BTL1010, Biospecs).
In-solution digestion was carried out for 10 µg of proteins from each condition. The samples were subjected to reduction and alkylation using 5 mM dithiothreitol (DTT) (60°C for 45 minutes) and alkylation using 10 mM Iodoacetamide (IAA). Trypsin (Gold mass-spectrometry trypsin; Promega, Madison, WI) digestion was carried out at 37°C for 10-12 hrs. The peptides were vacuum-dried and stored at -80°C until LC-MS/MS analysis.
[doi:10.25345/C5MB6T]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: proteomics, Lc-MS/MS,
Principal Investigators: (in alphabetical order) |
Dr. Amit Kumar Pandey, Translational Health Science and Technology Institute, India |
Submitting User: | drrenugoel |
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Owner | Reanalyses | |
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