Licor irdye 800cw goat anti rabbit igg h l

Cell cultures were lysed in ice-cold 1% NP-40 buffer (50 mM Tris HCl, 150 mM NaCl, 1% NP-40, pH 7.6) with protease inhibitor (Roche, Cat. No. 4693159001). Tissues were homogenized in ice-cold T-PER protein extraction reagent (Thermo Fisher Scientific, Cat. No. 78510) with protease inhibitor (Roche, Cat. No. 4693159001) using TissueLyser II (Qiagen). Total protein concentration was determined by bicinchoninic acid (BCA) method (Pierce, Cat. No. 23225). Normalized protein lysate was boiled with 4x Laemmli Sample Buffer (Bio-rad, Cat. No. 1610747) at 95 °C for 10 min. Primary antibodies rat anti-LAMP1 (BD Pharmingen, RUO - 553792) (1:2,000 dilution), mouse anti-FLAG M2 (Sigma, Cat. No. F1804) (1:5,000 dilution), rabbit anti-GAPDH (Abcam, Cat. No. ab9485) (1:5,000 dilution) and secondary antibodies LICOR IRDye 680RD Goat Anti-Rat IgG (H + L) (LI-COR Biosciences, Cat. No. 926-68076) (1:5,000 dilution), LICOR IRDye 680RD Goat Anti-Mouse IgG (H + L) (LI-COR Biosciences, Cat. No. 926-68070) (1:5,000 dilution), LICOR IRDye 800CW Goat Anti-Rabbit IgG (H + L) (LI-COR Biosciences, Cat. No. 926-32211) (1:5,000 dilution) were used in Western blot. Blot membrane was scanned with a LI-COR scanner (Odyssey). Western blot quantification analysis was performed with Image Studio Lite (LI-COR).