Recombinant protein a

Two monoclonal recombinant anti-GDob1 IgG3 allotypes, G3m(g) and G3m(s), were produced in an HEK-293F FreeStyle cell line expression system (Life Technologies, Paisley, UK). IgG3m(s) was then purified with a HiTrap MabSelect SuRE column packed with recombinant Protein A (GE Healthcare), while IgG3m(g) was purified with a Protein G HiTrap HP column (GE Healthcare).

Lysates were prepared from the brains of Tg6209 mice [15 (link)] and age-matched, non-transgenic animals, using either the protocol described above (three-step extraction protocol) or an alternative (one-step) protocol. For one-step extraction, each hemi-forebrain was mechanically dissociated in 1 mL buffer 3. The resulting homogenate was nutated for 1 h (4 °C), and then centrifuged (16,100×g, 90 min, 4 °C). Supernatants were then frozen for at least 24 h, then thawed and centrifuged (16,100×g, 90 min, 4 °C). Supernatants from this centrifugation step were depleted of endogenous immunoglobulins using Protein-G Fast Flow Sepharose beads, as described above. Protein concentrations were determined by a BCA protein assay kit (Thermo Scientific).
Additional lysates were prepared from the brains of hAPP-J20 mice [16 (link)] and age-matched, non-transgenic animals, using the three-step protocol described above. The brain extracts using buffer 1 were used for immunoaffinity purification. Lysates were depleted of endogenous immunoglobulins using Fast Flow Sepharose beads conjugated to native Protein A (Cat # 17528006, GE Healthcare Life Sciences), recombinant Protein A (Cat # 17127901, GE Healthcare Life Sciences), or Protein G.