Hippocampal Protein Extraction Protocol

Hippocampal samples were weighed and extracted using a 2-step extraction protocol with modifications as described previously (Youmans et al., 2012 (link); Tai et al., 2013 (link), 2014a (link); Thomas et al., 2016 (link), 2017 (link); Marottoli et al., 2017 (link), 2019 (link)), in order to separate out soluble and detergent-soluble proteins. Briefly, samples were homogenized using a bead mill (Fisherbrand) at 6 m/s for 1 cycle of 30 s in ice cold TBS at 10 μl/mg of brain tissue, centrifuged (100,000 × g for 30 min), and aliquoted. The resulting pellet was then resuspended in SDS buffer (1% SDS + 10 mM NaF + 2 mM Na₃VO₄ + 1× protease inhibitor cocktail in 20 mM HEPES; pH = 7.4), mixed via end-over-end rotation for 30 min at 4°C, sonicated (20% amplification, 3 cycles), centrifuged (100,000 × g for 30 min), and aliquoted. TBS and SDS buffer aliquots were flash frozen in liquid nitrogen and stored at −80°C. Total protein was quantified in TBS and SDS buffer extracts using the Pierce BCA Protein Assay Kit.