Novex 10 20 tris glycine gel

BN-PAGE and SDS-PAGE analyses were performed as described elsewhere [79 (link)] with some modifications. Briefly, for BN-PAGE, 5 µg sE2 was mixed with PBS and 4× loading dye [a mix of 500 µl 20× MOPS Buffer (1M MOPS+1M Tris, pH 7.7), 1000 µl 100 % glycerol, 50 µl 5 % Coomassie Brilliant Blue G-250, 600 µl milli-Q] and directly loaded onto a 4–12 % Bis-Tris NuPAGE gel (Thermo Fisher Scientific). The gels were run for 1 h at 200 V at 4 °C using NativePAGE Running Buffer (Invitrogen). BN-PAGE gels were stained using the Colloidal Blue Staining Kit according to the manufacturer’s instructions (Life Technologies). For SDS PAGE, 5 µg of sE2 was mixed with loading dye (25 mM Tris, 192 mM Glycine, 20 % v/v glycerol, 4 % m/v SDS, 0.1 % v/v bromophenol blue in milli-Q water) and incubated at 95 °C for 10 min prior to loading on a 4–12 % Tris-Glycine gel (Invitrogen). For reducing SDS-PAGE, dithiothreitol (DTT; 100 mM) was included in the loading dye and loaded on a Novex 10–20 % Tris-Glycine gel (Thermo Fisher Scientific). Gels were run in a buffer containing 25 mM Tris, 192 mM glycine and 0.5 % SDS for 1 h at 200 V at 4 °C. Coomassie blue staining of SDS-PAGE gels was performed using the PageBlue Protein Staining Solution (Thermo Fisher Scientific).

Purified sE2 monomers were treated with PNGase F or EndoH (New England BioLabs) following the manufacturer’s protocol. The samples were then run on a reducing Novex 10–20% Tris-Glycine gel (Thermo Fisher Scientific) and stained by Coomassie blue as described above. Hereafter, the samples from this same gel were transferred onto a nitrocellulose membrane. The blots were blocked in PBS+5% milk solution+0.1% Tween-20 and then probed by serial incubation with an anti-E2 antibody followed by a goat anti-human secondary conjugated to HRP. Chemiluminescence signal was then determined via the use of the Western Lightning Plus-ECL system (PerkinElmer).

SDS-PAGE analyses were performed as described elsewhere^{103 (link)} with some modifications. Briefly, 5 µg of E2E1 trimer were mixed with loading dye (25 mM Tris, 192 mM Glycine, 20% v/v glycerol, 4% m/v SDS, 0.1% v/v bromophenol blue in milli-Q water), and incubated at 95 °C for 10 min prior to loading on a 10–20% Tris-Glycine gel (Invitrogen). For reducing SDS-PAGE, dithiothreitol (DTT; 100 mM) was included in the loading dye and loaded on a Novex 10–20% Tris-Glycine gel (Thermo Fisher Scientific). Gels were run in a buffer containing 25 mM Tris, 192 mM glycine, and 0.5% SDS for 1 h at 200 V at 4 °C. Coomassie blue staining of SDS-PAGE gels was performed using the PageBlue Protein Staining Solution (Thermo Fisher Scientific).