Bca assay reagent

Prepare protein extracts from neutron-irradiated meat samples. 20 mM Tris–HCl (pH 7.5), 9 M Urea, and 2% CHAPS are used as extraction buffer. After crushing the sample in a bead crusher, centrifuge it (10,000 × g, 10 min, 10 °C) and collect the supernatant. Protein concentration of these extracts was determined by the bicinchoninic acid method (BCA assay reagent; Thermo Fisher Scientific, Rockford, IL, USA) using bovine serum albumin as a standard.

Whole-cell lysis from treated cells were extracted with RIPA buffer containing 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 140 mM NaCl. Lysis products were ultrasonicated and then centrifuged at 12,000 × g for 15 min at 4 ˚C. Protein concentrations were measured using BCA Assay Reagent (#23228, Thermo Fisher Science, Inc.). The western blotting was performed as described previously [15 (link)]. Protein samples (20 μg) were subjected to SDS-PAGE electrophoresis and IB analysis. Briefly, the proteins were transferred to a PVDF membrane and then incubated with 5% skimmed milk. Primary antibodies were incubated overnight at 4 ˚C, and secondary antibody was incubated for 30 min at room temperature. The immunoblot bands were observed with ECL reagents (#34579, Thermo Fisher Scientific, Inc.).

Whole-cell extracts were prepared using RIPA buffer containing protease and phosphatase inhibitor cocktails (87785, 78420; Thermo Scientific, Waltham, USA), and the protein concentrations were determined by using bicinchoninic acid (BCA) assay reagent (Thermo Scientific). Equal amounts of protein samples (30 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in PBS with 0.1% Tween-20 (1×PBST) containing 5% fat-free milk at room temperature for 2 h and then incubated with primary antibodies overnight at 4°C. Then, membranes were washed with 1×PBST for 3 times followed by 2 h of incubation with the corresponding HRP-conjugated secondary antibodies at room temperature. Membranes were washed with 1×PBST for 3 times and visualized using enhanced chemiluminescence kit (Invitrogen, Carlsbad, USA). Protein bands were analyzed using the Gel Doc XR ChemiDoc imaging system (Bio-Rad, Hercules, USA) and quantified using Quantity One software (Bio-Rad).

HepG2 cells were seeded in a 35-mm culture dish with a density of approximately 1 × 10⁶ cells/dish and allowed 24 h to attach. Cells were harvested and lyzed by M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) containing the proteinase inhibitor cocktail (Thermo Fisher Scientific, USA). Protein from cell lysate was collected and concentration quantified by BCA Assay Reagent (Thermo Fisher Scientific, USA). Equal amounts of proteins per lane were separated by 8–12% (SDS) polyacrylamide gel electrophoresis and transferred to PVDF membranes. Then, the membranes were incubated with RAPIDBLOCK solution (Thermo Fisher Scientific, USA). Membranes were then incubated with anti-FASN (Abcam, USA), anti-ACC (Merck Millipore, USA), and anti-ACLY (Cell Signaling Technology, USA) and then exposed to horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Life Technologies, Invitrogen). β-actin was used as an internal control (Cell Signaling Technology, USA). Finally, protein bands were visualized by LuminataTM Forte Western HRP Substrate (Merck Millipore, USA) and detected by CCD camera (Chemiluminescence Image Quant LAS 4000; GE Healthcare Life Sciences, Pittsburgh, PA, USA). Percentages of relative expression levels of protein/actin were calculated by Image J software version 1.46.

ECs were washed with cord buffer after treatment [NaCl (0.14 M), KCl (4 mM), glucose (11 mM), and HEPES (10 mM, pH 7.4)] and then lysed with 100 μL of lysis buffer [HEPES (250 mM, pH 7.7), EDTA (1 mM), neocuproine (0.1 mM), and CHAPS (0.4%, w/v)]. After centrifugation, protein supernatant was collected and protein concentrations were determined with BCA assay reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA).

Cells were scraped and lysed in ice-cold RIPA buffer (Thermo Scientific, Waltham, MA, USA) supplemented with protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA). After centrifugation, the supernatants were collected and a BCA Assay Reagent (Thermo Scientific) was used to determine protein concentrations. Fifty-microgram aliquots of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. After blocking with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 for an hour, bound proteins were exposed to the following antibodies overnight at 4°C: MET (#8198 rabbit monoclonal; Cell Signaling Technology), p-MET (#3077 rabbit monoclonal; Cell Signaling Technology), β-actin (sc-47778 mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Akt (#4691 rabbit monoclonal; Cell Signaling Technology), p-Akt (#4060 rabbit monoclonal; Cell Signaling Technology), Erk (#4695 rabbit monoclonal; Cell Signaling Technology), and p-Erk (#4370 rabbit monoclonal; Cell Signaling Technology). The secondary antibodies used were HRP-conjugated goat anti-rabbit and anti-mouse IgG (GE Healthcare, Little Chalfont, Buckinghamshire, UK). An ECL plus Western Blotting Detection System kit (GE Healthcare) was used to detect western signals.

Keratinocytes after treatment were washed with cord buffer [NaCl (0.14 M), KCl (4 mM), glucose (11 mM), and HEPES (10 mM, pH 7.4)] and then lysed with 100 μL of lysis buffer [Hepes (250 mM, pH 7.7), EDTA (1 mM), neocuproine (0.1 mM), and CHAPS (0.4%, w/v)]. After centrifugation, protein supernatant is collected and protein concentrations are determined with BCA assay reagent (Thermo Fisher Scientific Inc, Rockford, IL, USA).