Goat anti human pigr antibody

Protein samples were separated on a 10% SDS-polyacrylamide gel and then transferred to nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). Membrane was blocked with 5% skimmed milk in Tris-buffered saline with Tween (TBST) (25 mM Tris, 200 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) for 1 h at room temperature before incubation with primary antibody (goat anti-human PIGR antibody [R&D Systems 1:5000] or mouse anti-human α-tubulin antibody [Sigma-Aldrich 1:500]) overnight at 4 °C. After washing three times with TBST, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibody (rabbit anti-goat IgG HRP-conjugated antibody [R&D Systems 1:1000] or goat anti-mouse IgG HRP-conjugated antibody [Sigma-Aldrich 1:70,000]) for 1 h at room temperature. The membrane was washed thrice with TBST and enhanced chemiluminescent detection was performed using Immobilon Western chemiluminescent HRP substrate (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The chemiluminescent signal was visualized using a Luminescent Image Analyzer (Fujifilm Life Science, Cambridge, MA, USA).

Protein samples were separated on a 10% SDS–polyacrylamide gel and then transferred to nitrocellulose membrane. Membrane was blocked with 5% skimmed milk in TBST for 1 h at room temperature before incubation with primary antibody (goat anti-human PIGR antibody [R&D Systems, Minneapolis, MN, USA 1:500]) overnight at 4 °C. After washing 3 times with TBST, membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibody (rabbit anti-goat IgG HRP-conjugated antibody [R&D Systems 1:1000]) for 1 h at room temperature. The membrane was washed thrice with TBST and enhanced chemiluminescent detection was performed using Immobilon Western Chemiluminescent HRP substrate (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The chemiluminescent signal was visualized under Luminescent Image Analyzer (Fujifilm Life Science, Cambridge, MA, USA).