Biochemical characterization of LRRK2 and testing of LRRK2 antibodies on mouse tissue. A) Over-expressed mouse LRRK2 detected by JH5514 versus empty vector transfected HEK-293T cells. Whole mouse brain was mechanically homogenized in either PBS (low salt buffer), high salt buffer (PBS supplemented to 600 mM NaCl), 1% Triton X-100 (in PBS), 1% SDS (in PBS) or RIPA (1% SDS, 1% sodium deoxycholate, 1% NP-40). All buffers contained complete protease inhibitors (Roche). A knockout mouse brain is homogenized in PBS (low salt) alone and shown as control. B) Lysates derived from HEK-293T cells transfected with mLRRK2 plasmids were incubated in 1 × Laemmli sample buffer at the indicated temperatures for 10 minutes and then analyzed by SDS-PAGE. Plasticware used to process the protein samples were either siliconized or left untreated. Indicated samples were supplemented with 5% Formic acid (FA) or 4M urea after 10 minute incubation at the indicated temperature. C) LRRK2 antibodies Novus 267 and 268, Abgent AP7099b, and Chemicon (AB9682) in addition to Alexis (AT106) and Cell Signaling (2567) were tested on over-expressed mouse LRRK2 protein and wildtype and LRRK2-deficient mouse brain homogenized in PBS alone. All antibodies recognize cross-reactive bands near the expected size of LRRK2. An arrow denotes the position of LRRK2 in the Alexis (AT106) blot. All antibodies were tested on at least two independent membranes and lysates using optimized exposure conditions with similar results.
Biskup et al. BMC Neuroscience 2007 8:102 doi:10.1186/1471-2202-8-102