Odyssey blocking buffer pbs

The Western blotting analysis was performed as described previously^{40 (link)}. The samples were prepared by using 10 μg of total protein and subjected to 5–10% SDS-PAGE. Then, the separated proteins were transferred onto an Immobilon FL polyvinylidene difluoride membrane (Merck Millipore, Burlington, USA). The membrane was incubated in Odyssey® Blocking Buffer (PBS) (Merck Millipore, Burlington, USA) for 1 h at room temperature to block nonspecific binding and probed overnight with primary antibodies with the following dilutions: 1:1000 for the ABCG2 antibody and 1:2000 for the dynamin 2 antibody. After overnight incubation with primary antibody at 4 °C, the membrane was washed three times with 0.1% Tween 20-PBS (PBST) and treated with Alexa Fluor 680 anti-mouse IgG or anti-rabbit IgG antibody for 1 h at room temperature. Thereafter, the membrane was washed three times with PBST. The specific protein signals from the bound labeled antibodies were visualized with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, USA).

Proteins were denatured in XT sample buffer (cat. No. 1610791; Bio-Rad) and XT reducing agent (cat. No. 1610792; Bio-Rad), heated at 99°C for 10 min, and centrifuged for 5 min at 16,000g (room temperature). 25 μg of each protein mixture was separated by SDS–PAGE (polyacrylamide gel electrophoresis) on a Criterion XT 4–12% Bis-Tris gel (cat. No. 3450124; Bio-Rad Laboratories). Proteins were transferred to a PVDF membrane (cat. No. IPFL00010; Merck Millipore) after which the membrane was blocked using Odyssey blocking buffer (PBS) (cat. No. 927-4000; LI-COR) diluted once with TBS-T (TBS supplemented with 0.1% Tween-20). Immunoblots were incubated overnight with primary antibodies against GAPDH (ab8245; Abcam), HSP60 (sc-13115; Santa Cruz), lamin B (sc-374015; Santa Cruz), ribophorin I (sc-12164; Santa Cruz), or γ-tubulin (MA1-850; Thermo Fisher Scientific) in Odyssey blocking buffer (PBS) diluted once with TBS-T. Blots were washed four times with TBS-T and incubated with fluorescently labeled secondary antibodies (IRDye 800CW Goat Anti-Mouse IgG polyclonal 0.5 mg from cat. No 926-32210; LI-COR and IRDye 800CW Donkey Anti-Goat IgG polyclonal 0.5 mg from cat. No. 926-32214; LI-COR) in Odyssey blocking buffer diluted once with TBS-T for 1 h. After three washes with TBS-T and an additional wash in TBS, immunoblots were imaged using the Odyssey infrared imaging system (LI-COR).