Luminol

A Western blot of a patient serum pool and a control serum was performed to analyze the binding patterns to TE and deglycosylated TE. 10 μg of each protein extract was run on SDS-PAGE and blotted on to PVDF membranes as described above. The membranes were blocked for 2 hours by incubation with blocking buffer at RT and then incubated overnight with either a patient serum pool of sera from 8 patients (patients 16, 17, 20, 30, 31, 41, 42, and 46), or serum from control C6, both diluted 5 times in blocking buffer, or with blocking buffer alone for the secondary antibody control. The serum pool for patients with AGS had an IgE level to α-Gal of 83 kU_A/L and 5.2 kU_A/L to TE. For detection of proteins bound by the patient and control serum IgE, the membranes were incubated with secondary mouse-anti-human-IgE conjugated with horseradish peroxidase (clone B3102E8, Abcam), diluted 1:2,500. The protein bands were visualized with luminol (GE Healthcare) and evaluated in the Chemidoc instrument.

Samples from bacterial expression and subsequent purification steps were separated on 15% glycine SDS-PAGE or 15% tricine SDS-PAGE gels [66 (link)]. Where indicated, reduced samples were treated with 40 mM DTT. Protein bands were visualized with Coomassie Brilliant Blue, and images were recorded with a BioRad Chemidoc Imaging System. For western blotting, proteins separated by SDS-PAGE were transferred to a PVDF membrane (Immobilon-P, Merck Millipore) in a Mini Trans-Blot (Bio-Rad) transfer system. A mouse monoclonal His-tetra (Qiagen) antibody (1:1,000 dilution) was used in combination with a horseradish peroxidase–conjugated α-mouse secondary antibody (Pierce) (1:100,000 dilution). Chemiluminescence detection was performed using ECL Select Peroxide and Luminol solutions (GE Healthcare) according to the manufacturer’s directions.

To obtain protein extracts, HUVECs were scraped with lysis buffer (20 mM Hepes, pH 7.4, 1% Triton X-100, 100 mM NaCl, 50 mM NaF, 10 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)) and centrifuged for pellet (protein) recovery.
Western blotting (WB) was performed for the determination of protein relative levels: 10 μg of proteins were denatured using the sample buffer (Tris 40 mM, EDTA, bromophenol blue 0.01%, sucrose 40%, SDS 4%, and β-mercaptoethanol 10%) and heated to 95 ℃ for 5 min. Proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes (Whatman GmbH, Dassel, Germany). Proteins were incubated with specific primary antibodies, and detection was performed using their corresponding peroxidase-linked secondary antibodies (Supplementary Table S1). For loading control, β-actin was used. Finally, Luminol (ECL Western Blotting Detection Reagents, GE Healthcare, Hatfield, and Hertfordshire, UK) was added to the membrane for revealing proteins with an image reader LAS-4000 (GE Healthcare, Uppsala, Sweden). For signal density analysis, ImageJ software (NIH Image, National Institutes of Health, Bethesda, MD, USA) was used.