Western Blot Analysis
Whole protein extracts were diluted to concentrations of 1 mg/mL and
separated by SDS-PAGE under reducing conditions (2.5%
β-mercaptoethanol). Membranes were incubated with rabbit anti-Ara h 1,
Ara h 2 or Ara h 8 polyclonal antibody (1:1,000, 4°C overnight; Indoor
Biotechnologies, VA, USA). Bound antibodies were visualized using
horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG
monoclonal antibody (1:1,000, 1h room temperature; BioLegend, CA, USA)
and Clarity/Clarity Max enhanced chemiluminescence (ECL) substrates
(Bio-Rad Laboratories, Canada). Products were visualized with the
ChemiDoc XRS+ Imaging System (Bio-Rad Laboratories).