Protein a sepharose fast flow beads

As described in Ref. (20 (link)), IgG was isolated from plasma by affinity chromatography using 96-well protein G monolithic plates (BIA Separations, Ljubljana, Slovenia) for KORA F4 samples and Protein A Sepharose Fast Flow beads (GE Healthcare, Uppsala, Sweden) for the LLS samples. For the KORA F4 sample analysis, 100 µL of plasma was first diluted 10× with 1× PBS and then filtered through 0.45 µm GHP filter plate (Pall Corporation, Ann Arbor, MI, USA). Following, it was applied to the protein plate and instantly washed. With 1 mL of 0.1 M formic acid (Merck, Darmstadt, Germany), the IgGs were eluted from the protein plate and neutralized with 1 M ammonium bicarbonate (Acros Organics, NJ, USA). For the LLS sample analysis, 2 µL of plasma was incubated together with 15 µL of Protein A beads in a total volume of approximately 180 µL PBS in 96-well filter plates. The samples were then washed thrice with PBS and thrice with MilliQ-purified water, before elution with 0.1 M formic acid (Fluka, Steinheim, Germany). The samples were subsequently dried in a vacuum concentrator for 2 h at 60°C.
Due to the different IgG isolation procedures for KORA F4 and LLS, we obtained subclass-specific measurements for IgG1, IgG2/IgG3, and IgG4 in KORA F4 and IgG1, IgG2, and IgG4 in LLS. IgG3 is less abundant compared with IgG2 and we thus denote the IgG2/IgG3 mixture in KORA as IgG2 only.

IgGs were purified in 96-well format as described previously^{18 (link)}. An amount of 2 µL of plasma was incubated with 15 µL Protein A Sepharose Fast Flow beads (GE Healthcare, Uppsala, Sweden) and 150 µL phosphate buffered saline (PBS) on a 96-well filter plate for 1 hour at room temperature while shaking. The IgG samples were washed three times with PBS and three times with MilliQ-purified water, followed by elution with 100 µL 100 mM formic acid. The samples were then dried in a vacuum concentrator for 2 hours at 60 °C and resuspended in 40 µL 25 mM ammonium bicarbonate with 200 ng of trypsin (sequencing grade modified trypsin, Promega, Madison, WI). Digestion took place overnight at 37 °C. Two of the 96-well plates were prepared twice to assess interbatch variation.

Cells were lysed in HEPES lysis buffer [50 mM HEPES, 100 mM sodium chloride, 50 mM sodium fluoride, 50 mM sodium β-glycerophosphate, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1 mM sodium orthovanadate, 100 nM okadaic acid, protease inhibitors (Roche Molecular Biochemicals) and 1% Nonidet P-40]. One milligram of cleared lysate was added to 10 µl of Protein A Sepharose fast flow beads (GE Healthcare) covalently coupled with antibodies against MKK3, MKK4 or MKK6, and incubated for 3 h at 4°C. Beads were washed three times with HEPES lysis buffer followed by elution using 10 µl of non-reducing Laemmli buffer and immunoblotting. Immunoblotting of cell lysates was carried out as described previously [11 (link)].