Streptavidin coated magnetic particles

Affinity purification of agAAb was done by a biotinylated peptide biotin-AINCYANETCCD corresponding to the second extracellular loop of the β2AR. After incubation of 1 ml IgG with 300 μl of the peptide (100 μg/ml; for 1 h), this procedure was followed by an incubation with streptavidin-coated magnetic particles (Roche, Germany; for 30 min). The separation was performed with a magnetic separator (Dynal, Germany). After washing with PBS, eluation with 3 M KSCN in two 0.5 ml steps was done. Afterwards the agAAb were dialyzed with PBS (48 h, 4°C).

Cells were extracted with a lysis buffer (50 mM Tris-HCl pH8.0, 100 mM NaCl and 0.5% IGEPAL CA-630) supplemented with the above-mentioned protease inhibitors. Extracts were incubated for 1 h at 4 °C with streptavidin-coated magnetic particles (Roche, Basel, Switzerland). Bound proteins were digested using the FASP protocol and then analyzed via LC-MS/MS.

IgG preparations of patients with POAG and OHT were affinity purified using the biotinylated peptide biotin-AINCYANETCCD corresponding to the second extracellular loop of the β2AR. One milliliter of IgG was treated with 300 μl of the peptide (100 μg/ml) for 1 h. The peptide–antibody complex was incubated with streptavidin-coated magnetic particles (Roche, Germany) for 30 min. The separation was performed with a magnetic separator (Dynal, Germany). The particles were washed five times with PBS. The antibodies were eluted with 3 M KSCN in two 0.5 ml steps. The antibody solution was dialyzed against PBS for 48 h at 4°C.