SDS-PAGE and western blotting
Four microliters of raw or cooked fish extracts were separated on a
13.5% SDS-polyacrylamide gel according to their molecular weights using
a Mini PROTEAN SDS-PAGE system (Bio-Rad). Protein bands were stained
with SimplyBlue SafeStain (Thermo Fisher). Western blotting was
performed using the surf-blot antibody system (Idea Scientific). One
hundred fifty microliters of raw fish extracts were resolved in 13.5%
SDS-PAGE and transferred to PVDF membranes (Bio-Rad) with the Trans-Blot
Turbo system (Bio-Rad). Non-specific binding of the membranes was
blocked with 5% non-fat dry milk in 0.05% Tween-20 in Tris-buffered
saline (TBS-T, blocking solution) for 1 hour at room temperature.
Patient sera diluted at 1:10, 1:20 or 1:30 (adjusted for sIgE levels)
and HRP-conjugated anti-human IgE (Southern Biotech) diluted at 1:2000
in blocking solution were used for probing IgE-binding proteins.
IgE-binding fish proteins were visualized by incubating the membranes
with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo
Fisher), and the signal was acquired with the ChemiDoc MP Imaging System
(Bio-Rad).