*4.3. Preparation of FLAG-Peptide-AntiFLAG Antibody Immune Complex-Containing Solution*

A volume of 20 µL of 1 µg/µL of mouse monoclonal antiFLAG M2 antibody (product code F 1804, Sigma-Aldrich, Steinheim, Germany) was first re-buffered into 200 mM ammonium acetate buffer, pH 7, using a centrifugal filter (Amicon Ultra cutoff 50 K; Merck Millipore Ltd., Tullagreen, Carrigtwohill Co Cork, Ireland), as described [10,13]. After buffer exchange, 5 µL of antiFLAG antibody solution (0.2 µg/µL; 1.33 µM) was mixed with 1.5 µL of a peptide mixture of seven peptides containing 10 µM each of GPI peptide (ALKPYSPGGPR, 1141.62 Da), FLAG peptide (DYKDDDDK, 1012.40 Da), Angiotensin II (DRVYIHPF, 1045.53 Da), TRIM21A peptide (LQELEKDEREQLRILGE, 2097.11 Da), TRIM21B peptide (LQPLEKDEREQLRILGE, 2065.12 Da), TRIM21C peptide (LQELEKDEPEQLRILGE, 2038.06 Da), and RA33 peptide (MAARPHSIDGRVVEP-NH2, 1632.86 Da) in a molar ratio of 2.2:1 of peptide to antibody. Solvent for the peptides was 200 mM ammonium acetate buffer, pH 7.
