Piece bca protein assay

Snap frozen hippocampal tissues were hand homogenized in Pierce RIPA buffer (Thermo Scientific, #89900) with cOmplete ULTRA tablets (Roche, #05892791001) and PhoSTOP (Roche, #04906837001) protease inhibitors for whole cell lysis. Debris was removed via centrifugation and the protein concentration of the remaining homogenate was measured using the Piece BCA Protein Assay (Thermo Scientific, #23225) following the manufacturer’s instructions. 20 ug of protein in Laemelli buffer (BioRad, #1610747) with beta mercaptoethanol (Sigma, M–3148) was boiled at 100 °C, loaded into a precast 4–15% Tris Glycine gel (BioRad, #5671084), and run using Tris/Glycine/SDS buffer (BioRad, 1610772) at 150 V. Protein was transferred onto a nitrocellulose membrane (BioRad, 1620168) using Tris/Glycine buffer (BioRad, 1610771) at 100 V. Membranes were blocked using Odyssey Blocking Buffer (PBS) (LI-COR, 92740000) and incubated for a minimum of 14 h with either synapsin 1 (1:2000 in PBS, Millipore, #AB1543) or PSD95 (1:1000 in PBS, Abcam, #ab13552). Blots were washed using TBS and incubated for 60 min in their host-specific secondary antibody (LI-COR, #92632210-Mouse, #92632211-Rabbit). No bands were visible when secondary antibodies alone were added. Blots were imaged using Odyssey (LI-COR) imaging system and quantified using the Image Studio software. All blots were normalized to GAPDH (Sigma, #G8795).