Unveiling Kounis Syndrome Induced by Mustard Protein (Sin a 2) in an Adult With Lipid Transfer Protein Syndrome: A Novel Case Report

Given the lack of identification of mustard proteins, the samples were sent to the Biochemistry and Molecular Biology Department (Complutense University, Madrid) in order to identify the protein. Protein extractNatural seeds were crushed under liquid nitrogen and the flour was delipidated in cold acetone (Scharlab).Sediment was air-dried, resuspended in 20 mM ammonium bicarbonate, pH 6.5, and homogenized using an iced potter for protein extraction.Total protein concentration was quantified through Bradford assay. Electrophoresis and Western BlotSDS-PAGE was carried out in 17% polyacrylamide gel on non-reducing conditions, loading 20 g of total protein per well.After electrophoresis, proteins were stained with Coomassie Blue R-250 or, for Western Blot development, they were transferred to nitrocellulose membranes (Hybond, Amersham).WB was carried out after membrane blocking with PBS-Tween20 (0.1% (v/v)) -1.5% (w/v) delipidated powder milk O.N. 4C.Membrane incubation was performed with patient serum (dilution 1/20) on PBS-Tween20 0.05% -1.5% delipidated powder milk for 2 hours R. T., followed with washing steps (PBS-Tween20 0.1%).Secondary antibody (IgE-HRP) was incubated on the membrane for 1 hour R.T. diluted 1/2000 on PBS-Tween20-milk (as previous).After membrane washing, band detection was conducted using ECL Western Blotting Substrate (Thermo Scientific), and images were captured at different exposition times in a ChemiDoc XRS+ system (Bio-Rad).