The purified viruses (about 100 μg HA protein) were mixed with Pierce IP lysis buffer (Thermo-Fisher, San Jose, CA) and Halt Protease Inhibitor Cocktail (Thermo-Fisher, San Jose, CA).24 (link) The viral membrane lysis was conducted according to the manufacturer’s instructions. The supernatant was collected after centrifugation, and the protein concentration was determined by Quant-iT Protein Assay Kit (Thermo-Fisher, San Jose, CA). The samples were then suspended in 100 μL of 100 mM ammonium bicarbonate buffer (Alfa Aesar, Tewksbury, MA) and 2,2,2-trifluoroethanol (TFE) (Sigma-Aldrich, St. Louis, MO). After suspension, the samples were then digested in trypsin and cleaned up as aforementioned.
Protocol detail
Viral Membrane Lysis and Protein Digestion
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Pierce IP lysis buffer
Halt Protease Inhibitor Cocktail
Quant-iT Protein Assay Kit
2,2,2-trifluoroethanol (TFE)
Ammonium bicarbonate
Assay
Buffer
Centrifugation
Membrane
Protease inhibitor
Protein
Trifluoroethanol
Trypsin
Viruses
Corresponding Organization : University of Missouri
Other organizations : Boston University, Georgia State University
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Variable analysis
independent variables
- Purified viruses (about 100 μg HA protein)
- Protein concentration
- Pierce IP lysis buffer (Thermo-Fisher, San Jose, CA)
- Halt Protease Inhibitor Cocktail (Thermo-Fisher, San Jose, CA)
- 100 mM ammonium bicarbonate buffer (Alfa Aesar, Tewksbury, MA)
- 2,2,2-trifluoroethanol (TFE) (Sigma-Aldrich, St. Louis, MO)
- Trypsin digestion
- No positive or negative controls were explicitly mentioned.
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