Ecl western blotting reagent

SDS-PAGE gels were prepared using the Bio-Rad protein electrophoresis systems. Zebrafish embryos were homogenised in RIPA (50 mM Tris-HCl, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 1 mM EDTA, 0.1% (w/v) SDS) lysis buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich) using a syringe and needle. Supernatants were collected after a brief centrifugation and boiled in 4X loading buffer (200 mM Tris-HCl (pH 6.8), 400 mM DTT, 8% SDS, 0.4% bromophenol blue and 40% glycerol) before loading. After transfer of proteins, membranes were rinsed with TBSTw (Tris Buffered Saline, 0.1% Tween-20) once and blocked in 5% skimmed-milk powder in TBSTw for 1 hour before incubation with primary antibody overnight. After incubation, membranes were rinsed 4X in TBSTw for 5 minutes and transferred to secondary antibody for 4 hours at room temperature, excess antibody subsequently removed by a further 4 washes in TBSTw for 5 minutes before detection with ECL Western blotting reagent (Bio-Rad) following manufacturer’s instructions. Signal detection was performed using a ChemiDoc MP Imaging system (Bio-Rad) or with CL-XPosure Film (34089, Thermo Fisher Scientific).

Aliquots containing 100 ng Ca²⁺-bound monomers and amyloids of Gad m 1 chains were spotted in duplicate on a nitrocellulose membrane. Immunodetection was performed by 1 h of incubation with anti-amyloid fibril OC (AB2286 Merck Millipore, 1/2000 dilution) and anti-amyloid oligomer A11 (AB9234 Merck Millipore, 1/2000 dilution) antibodies, followed by extensive washes and 30 min of incubation with horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000 diluted; Sigma-Aldrich) [25 (link),26 (link)]. ECL Western blotting reagent (BioRad) and a ChemiDoc XRS instrument (BioRad) were used for signal development and detection, respectively.

MDCK cells were collected and lysed in CO-IP lysis buffer for 40 min. Then, the cells were centrifuged at 12,000 × g for 10 min at 4 C. Proteins were analysed by SDS-PAGE and then transferred to PVDF membranes (Bio-Rad, California, United States). Five percent milk solution was used to block the membrane at room temperature for 1 h, and then the membrane was incubated with the primary antibody overnight at 4 C. The protein signal was visualized using ECL Western blotting reagent (Bio-Rad, California, Unites States). The NP antibody and NA antibody used to detect influenza virus and the antibody to detect GAPDH were all purchased from GENE Tex (Southern California, Unites States).