Te77 semi dry transfer unit

Protein samples were run on an SDS-PAGE gel alongside a pre-stained ladder (Invitrogen, Waltham, U.S.) and then transferred onto a nitrocellulose membrane (Bio Rad Laboratories, Hercules, U.S.) in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% (v/v) methanol, pH 8.3) using a TE77 semi-dry transfer unit (Hoefer, Holliston, U.S.) at 200 V, 50 mA/gel for 1 h. The membrane was incubated by blocking solution (TBS-T plus 5% (w/v) skim milk powder) on a shaker at RT for at least 1 h to block nonspecific binding sites of the membrane. Following this step, the membrane was washed twice with TBS-T (20 mM Tris-Cl, 150 mM NaCl, pH 7.6, 0.1% (v/v) Tween-20) for 5 min. The membrane was probed with human sera from patients with invasive GAS infections and healthy donors at 1/100 dilution in probing solution (TBS-T plus 2.5% (w/v) skim milk powder) for 1 h at RT. The membrane was then washed three times with TBS-T for 5 min each and incubated with HRP-conjugated goat anti-human IgG (Abcam) in a probing buffer and incubated for 1 h followed by washing with TBS-T three times for 5 min. Western blots were developed with ECL detection reagent (Amersham Biosciences) and analyzed using a ChemiDoc^TM imaging system (BioRad).

ILY (0.85 μM) was incubated with or without liposomes (0.29 mg/ml) and with or without ^cytoCD59 (0.85 μM) for 1 hour at 37 °C. Samples were analyzed by SDS-AGE using a 2% (w/v) gel (100 V, 1 hour). The proteins were transferred to a nitrocellulose paper using a TE77 Semi-dry transfer unit (Hoefer) at 70 mA for 2 hours at 4 °C. His-tagged ILY was detected using a Penta-His antibody (Qiagen), followed by a Anti-Mouse IgG -Alkaline Phosphatase (Sigma). Band visualization was carried out using Sigma Fast BCIP/NBT tablets (Sigma).