Pierce nhs activated magnetic beads

Commercial NHS-functionalized superparamagnetic beads (Pierce™ NHS-activated magnetic beads, ref 88826, ThermoFisher Sci., Les Ulis, France) were coupled to goat anti-rIgG according to the manufacturer’s instructions. In brief, magnetic beads’ slurry (150 μL) was pipetted into a 1.5 mL microtube. Using a magnet stand, magnetic beads were collected and the supernatant discarded. Then, ice-cold 1 mM hydrochloric acid (0.5 mL) was added and gently mixed. The magnetic beads were collected and the supernatant discarded. Goat anti-rabbit IgG solution (1 mg/mL in 50 mM borate pH 8.5; 150 μL) was added and incubated for 2 h under rotation. Magnetic beads were collected and 0.1 M glycine (pH 2.0; 0.5 mL) was added and mixed well. The magnetic beads were collected and the supernatant discarded. Then, 3 M ethanolamine (pH 9.0; 0.5 mL) was added, mixed well and incubated for 2 h under rotation. Magnetic beads were washed once with deionized water (0.5 mL) and twice with borate buffer (0.5 mL). Finally, MB-Ab conjugate was stored at 4 °C in 50 mM borate pH 8.5 (150 μL; final concentration 10 mg/mL) until use.

To isolate anti-chitin immunoglobulins, 50 µl of chitin magnetic beads (NEB E8036) were washed with 1 × PBS tween-20 (0.05%) three times and incubated with 200 µl of human serum rotating overnight at 4 °C. The chitin beads were washed four times with 1 ml of 1 × PBS tween-20 (0.05%) and eluted with 50 µl of 50 mM glycine–HCl (pH 2.7) for 30 s. The eluted sample was immediately neutralized with 5 µl of 1 M Tris-Base (pH 8) and run on the NCFGv1 microarray. To isolate immunoglobulins against the isoForssman antigen, 100 µl of a 100 µM solution of AEAB labeled glycan was coupled to 50 µl of Pierce NHS-Activated Magnetic Beads (ThermoFisher Scientific). After coupling, the beads were blocked with 3 M ethanolamine (pH 9.0) for 2 h, rotating at room temperature. Isolation of the anti-isoForssman antibodies was done as described above.

For pull-down assay, 0.1 mg/mL of purified protein was immobilised on Pierce NHS-activated magnetic beads (Thermo Fisher) following the manufacturers’ instructions. The beads were incubated with 10 mM EDTA and 50 μL serum (21 °C, 30 min). The beads were washed 3 times with 1 mL PBS + 0.05% (v/v) Tween20, once with 100 μL PBS, and boiled in 50 μL SDS-PAGE loading buffer. Elutions were analysed by SDS-PAGE and semi-dry Western blotting.

AGEs were removed from diabetic serum using an immunoadsorption method. To immunoprecipitate AGE-modified proteins, 500 μl of diabetic serum was incubated for 1 h with 25 μl of Pierce NHS-activated magnetic beads (Thermofisher Scientific) covalently conjugated with 10 μg of anti-AGE antibody (Abcam, see Supplementary Table S1 for antibodies in Additional file 1), according to the manufacturer instruction. To confirm the efficiency of AGE depletion, AGE concentration in both treated (unbound serum fraction) and untreated diabetic serum was evaluated in triplicate by ELISA (OxiSelect™ Advanced Glycation End-Product Competitive ELISA Kit, no. STA-817, Cell Biolabs, Inc., San Diego, CA, USA). Following this procedure, the concentration of AGEs in diabetic serum was reduced by about 60%, reaching a concentration similar to that of the non-diabetic serum (see the “Results” section).