Purification of GFP-tagged SHANK Proteins

Full-length rat GFP-Shank1A was already described (Romorini et al., 2004 (link)), as well as full-length rat GFP-tagged Shank2 (Boeckers et al., 2005 (link)) and full-length rat GFP-Shank3a (Grabrucker et al., 2011a (link)). HEK293T cells were cultivated in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C in 5% CO₂. For transient transfection, cells were plated on 10-cm petri dishes and cultivated until 70–80% confluence and were then transfected with either full-length rat GFP-Shank1A, rat GFP-tagged Shank2 or rat GFP-Shank3a. Therefore, an appropriate amount of DNA was mixed with DMEM with 10% FBS and PEI MAX 40K (Polysciences) transfection reagent and spread onto the cells. The cells were lysed 24 h after the transfection (lysis buffer of the μMACS GFP Isolation Kit (Miltenyi Biotec) + PhosphoSTOP and Complete Mini EDTA-free Protease Inhibitor Cocktail (both Roche) and purification of the recombinant GFP-tagged SHANK1, 2, and 3 proteins was performed using the μMACS GFP Isolation Kit (Miltenyi Biotec) according to manufacturer’s instructions. Purified recombinant proteins were stored at –20°C until usage.