S. aureus PSM solutions were prepared by diluting 1:100 with water from a 10 mg/ml stock in DMSO. PSM samples were incubated with filtered ThT at a final concentration of 0.2 mM.
PSMα3 and derivatives in powder form were treated with trifuoroacetic acid/hexafluoroisopropanol (1:1), dried in a chemical hood for 2 days, followed by further drying in a rotary vacuum concentrator for 2 h. Then, peptides were dissolved in 10 mM sodium phosphate buffer (pH 8.0) containing 150 mM NaCl. After sonication for 10 min, undissolved material was removed by centrifugation at 10,000 rpm for 5 min in a table top microcentrifuge. Alternatively, PSMs were diluted from 10 mg/ml stocks in DMSO in the same buffer and treated accordingly.
Thioflavin T (ThT) was purchased from AnaSpec (SensoLyte Thioflavin T β-Amyloid (1 – 42) Aggregation Kit). In each reaction, 200 µM peptide was mixed with 50 µM ThT in 10 mM sodium phosphate buffer (pH 8.0) and 150 mM NaCl. The fluorescence of ThT was scanned from 450 nm to 700 nm with excitation at 438 nm at the indicated times using a Tecan Safire multimode microtiter plate reader every 5 min at 37 °C.
PSMα3 and derivatives in powder form were treated with trifuoroacetic acid/hexafluoroisopropanol (1:1), dried in a chemical hood for 2 days, followed by further drying in a rotary vacuum concentrator for 2 h. Then, peptides were dissolved in 10 mM sodium phosphate buffer (pH 8.0) containing 150 mM NaCl. After sonication for 10 min, undissolved material was removed by centrifugation at 10,000 rpm for 5 min in a table top microcentrifuge. Alternatively, PSMs were diluted from 10 mg/ml stocks in DMSO in the same buffer and treated accordingly.
Thioflavin T (ThT) was purchased from AnaSpec (SensoLyte Thioflavin T β-Amyloid (1 – 42) Aggregation Kit). In each reaction, 200 µM peptide was mixed with 50 µM ThT in 10 mM sodium phosphate buffer (pH 8.0) and 150 mM NaCl. The fluorescence of ThT was scanned from 450 nm to 700 nm with excitation at 438 nm at the indicated times using a Tecan Safire multimode microtiter plate reader every 5 min at 37 °C.