Goat anti rabbit horseradish peroxidase linked antibodies

Aliquots (15 μL) of 50% (w/w) honey solution were resolved by SDS-PAGE using a Mini-Protean II electrophoresis cell (Bio-Rad). The proteins were transferred onto a 0.22-μm nitrocellulose Advantec membrane (Sigma-Aldrich) in 12 mM Tris, 95 mM glycine and 20% methanol using the wet blotting procedure. The membrane was blocked for 1 h in a Tris-buffered saline-Tween (TBST) buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 0.05% Tween 20) containing 5% non-fat dried milk and incubated overnight with a rabbit polyclonal antibody against honeybee GOX (1:2000 in TBST) which was prepared by GenCust Europe (Dudelange, Luxembourg). After washing with TBST, the membranes were incubated for 2 h in blocking buffer containing goat anti-rabbit horseradish peroxidase-linked antibodies (1:2500 in TBST; Promega). Immunoreactive bands were detected in solution containing dissolved SigmaFast 3,3-diaminobenzidine tablets (Sigma-Aldrich), and specific bands were quantified by densitometry using ImageJ software (NIH, version 1.52a, Bethesda, MD, USA).

Aliquots (15 μl) of 50% (w/v) honey solution with or without proteinase K were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 16.5% Tricine-SDS-PAGE using a Mini-Protean II electrophoresis cell (Bio-Rad). The proteins were transferred onto a 0.22-μm nitrocellulose Advantec membrane (Sigma-Aldrich) in 48 mM Tris, 39 mM glycine and 20% methanol using the semi-dry blotting procedure. The membrane was blocked for 1 h in a Tris-buffered saline-Tween (TBST) buffer (50 mM Tris–HCl, pH 7.5, 200 mM NaCl, and 0.05% Tween 20) containing 5% non-fat dried milk and incubated overnight with a rabbit polyclonal antibody against honeybee GOX or Def-1 (1:2000 in TBST). After washing with TBST, the membranes were incubated for 2 h in blocking buffer containing goat anti-rabbit horseradish peroxidase-linked antibodies (1:2500 in TBST; Promega). Immunoreactive bands were detected in solution containing dissolved SigmaFast 3,3-diaminobenzidine tablets (Sigma-Aldrich) or detected using enhanced chemiluminescence Immobilon Western kit (Millipore, MA, USA).

The GOX content was determined semi-quantitatively according to Bucekova et al. (2019) [7 (link)]. Briefly, aliquots (15 μL) of 50% (w/w) honey solution were resolved by SDS-PAGE and the proteins were transferred onto a 0.22-μm nitrocellulose Advantec membrane (Sigma-Aldrich, Germany) using the wet blotting procedure. The membrane was blocked for 1 h in a Tris-buffered saline-Tween (TBST) buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl and 0.05% Tween 20) containing 5% non-fat dried milk and incubated overnight with a rabbit polyclonal antibody against honey bee GOX (Clone No. RB3232; 1:2000 in TBST), which had been prepared by GenCust Europe (Dudelnag, Luxembourg). After washing with TBST, the membranes were incubated for 2 h in blocking buffer containing goat anti-rabbit horseradish peroxidase-linked antibodies (1:2500 in TBST; Promega, USA). Immunoreactive bands were detected in a solution containing dissolved SigmaFast 3,3-diaminobenzidine tablets (Sigma-Aldrich), and specific bands were quantified by densitometry using the ImageJ software (NIH, USA).
Total proteins in bee-processed syrups were measured using the Quick Start Bradford protein assay (Bio-Rad, USA) as described in the instruction manual.