Nupage 10 bis tris protein gel

Western blotting was performed essentially as previously described (Blackwood et al., 2018 (link)). In brief, soluble protein lysates were prepared in solutions that contained 1x NuPage LDS Sample Buffer (Thermo Fisher Scientific, Waltham, MA, United States), and 1% β-Mercaptoethanol (Sigma, St. Louis, MO, United States). Samples were then boiled and resolved using NuPage 10% Bis-Tris Protein Gels (Thermo Fisher Scientific, Waltham, MA, United States). Proteins were electrophoretically transferred onto Trans-Blot^® Turbo^TM Midi Nitrocellulose membranes using the Trans-Blot^® Turbo^TM system (Bio-Rad, Hercules, CA, United States). Primary rabbit antibodies used were anti-OPRM1 (1:5000, ab17934), anti-OPRK1 (1:10000, ab183825), anti-OPRD1 (1:1000, ab176324), and anti-Cyclophilin B (CYPB) (1:10000, ab16045) purchased from Abcam (Cambridge, MA, United States). Secondary antibodies used were goat anti-rabbit (1:500, Sc-1404) conjugated HRP purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Following secondary antibody incubation, ECL clarity (Bio-Rad, Hercules, CA, United States) was used to detect gel bands on ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, United States), and intensities were quantified with Image Lab version 6.0 (Bio-Rad, Hercules, CA, United States) software.

Immunoblotting (IB) was performed as we previously described [25] (link), [26] (link), [27] (link), [28] (link). Briefly, the Blot Mini Gel Tank and Blot Module Set (Thermo) with precast polyacrylamide gels of NuPAGE 10% Bis−Tris protein gels (1.5 mm × 10 wells, Thermo) were used. Primary antibodies included rabbit anti-ISG15 mAb (Sinobiological, Beijing, China; 1:2000), rabbit anti-syntenin mAb (Abcam, Shanghai, China; 1:1000), rabbit anti-TSG101 mAb (Merck, Guangzhou, China; 1:2000), rabbit anti-CD9 pAb (Abcam; 1:2000), and rabbit anti-GRP78/BIP pAb (Proteintech, Wuhan, China; 1:2000). HRP-conjugated anti-rabbit IgG mAb (CST; 1:20000) was used as the secondary antibody.

Cells were lysed in ice with RIPA buffer (150 mmol/l Tris–HCl pH 7.0, 150 mmol/l NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1%SDS) containing with protease inhibitor cocktail (Roche, 4,693,159,001) for 30 min. The Bio-Rad protein assay was used to quantify the lysates. The protein samples were subjected to NuPAGE™ 10% Bis–Tris Protein Gels (Invitrogen, NP0302BOX) and probed with the indicated antibodies to visualize the protein levels using a ChemiScope 6000 Touch.

For SDS-PAGE, 1 µg of the hard protein corona samples were mixed with LDS reducing agent and sample buffer and incubated for 10 min at 70 °C. The samples were then applied on a NuPAGE 10% Bis-Tris protein gel (Thermo Fisher Scientific) and run for 1 h at 100 mV. The protein bands were stained using a SilverQuest Silver Staining Kit (Thermo Fisher Scientific) following the manufacturer instructions.