Trans blot turbo rta mini lf pvdf transfer kit

Proteins, isolated from the liquid nitrogen-preserved liver tissue using MicroRotofor™ Cell Lysis Kit, were quantified by Bradford assay using Quick Start™ Bovine γ-Globulin Standard. The protein extracts were mixed with 4× Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) (1,1) and heated at 95 °C for 5 min immediately before loading. The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). Tnfα levels were evaluated by Western-blot analysis using Trans-Blot® Turbo™ RTA Mini LF PVDF Transfer Kit Bio-Rad Laboratories, Inc. (Hercules, CA, USA) for protein transfer. Other Bio-Rad products used in the assay include Immun-Star Goat Anti-Rabbit (GAR)-HRP Conjugate as secondary antibodies, Clarity™ Western ECL with ChemiDoc™ system for signal development, and Image Lab™ software for data analysis.
The membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TTBS) for 1 h at room temperature and subsequently incubated with primary antibodies to Tnfα or β-tubulin (Abcam, Cambridge, UK) applied in 1:100 dilutions, as recommended by the manufacturer. The full-length images of blots are included in a Supplementary Information file. All blots were processed in parallel.

Total cell lysates were extracted using lysis buffer [Tris-HCl (pH 7.4), sodium dodecyl sulfate (SDS), mercaptoethanol, and glycerol]. Protein concentrations were determined using Takara Bradford Protein Assay kit (T9310A; Takara Bio, Shiga, Japan), and an equal amount of protein (10 µg) was separated on a 4–20% gradient SDS-PAGE (TEFCO, Tokyo, Japan). Proteins were transferred using a Trans-Blot Turbo RTA Mini LF PVDF Transfer kit (170-4274; Bio-Rad Laboratories, Hercules, CA, USA) to a Trans-Blot Turbo Mini-size LF polyvinylidene difluoride membrane (Bio-Rad Laboratories) and blocked with 5% non-fat dry milk in Tris-buffered saline [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween-20] for 1 h at room temperature. Membranes were then incubated with anti-KHSRP and anti-myc primary antibodies at dilutions of 1:1,000 in 5% non-fat dry milk in Tris-buffered saline overnight at 4°C. Following incubation with appropriate secondary antibodies conjugated with horseradish peroxidase (sc-2004, sc-2005; Santa Cruz Biotechnology) at dilutions of 1:2,000 for 1 h at room temperature, immunoreactive bands were visualized using the ECL Plus kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions.