Coomassie brilliant blue protein assay kit

The rats used for the high-molecular-weight contrast studies were subjected to biochemical analysis. Brain tissue was homogenized in lysis buffer (50 mM Tris, pH 7.4, 40 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na₃VO₄, 50 mM NaF, 10 mM sodium pyrophosphate, and 10 mM sodium-glycerophosphate supplemented with protease inhibitor cocktail) on ice for 30 min and then centrifuged at 10,000 rpm for 10 min at 4°C. Protein concentration from the supernatant was determined using a Coomassie brilliant blue protein assay kit (Bio-Rad). The same amount of supernatant was boiled in the SDS loading buffer. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were then blocked in 5% non-fat milk and incubated at 4°C overnight with a primary antibody. The primary antibodies used were polyclonal anti-beta amyloid protein (anti-Aβ, 27320-1-AP, ProteinTICh, USA) and rabbit anti-phosphorylated tau (AF6141, Affinity Biosciences, USA). Blots were incubated with HRP-conjugated secondary antibodies (S0001, Affinity Biosciences, USA) for 1 h at room temperature. Immunoreactive bands were visualized with a ChemiDoc imaging system (Bio-Rad).

Mouse brain tissue samples were lysed in lysis buffer (50 mM Tris, pH 7.4, 40 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium-glycerophosphate, supplemented with protease inhibitors cocktail) on ice for 30 min. For the in vitro experiment, cells were lysed in RIPA buffer (pH 7.5, 20 mM Tris–HCl, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) on ice for 30 min. The lysates were centrifuged for 10 min at 15,000 rpm. We quantified proteins in the supernatant using a Coomassie Brilliant Blue protein assay kit (Bio-Rad). Next, we boiled the supernatant mixed with the same amount of SDS loading buffer. After SDS- PAGE, the samples were transferred to a nitrocellulose membrane. The membranes were blocked in 5% non-fat milk for 1 h at room temperature, then incubated with primary antibody at 4°C overnight. Then the blots were incubated with IRDye 800CW-conjugated affinity-purified anti-mouse or anti-rabbit IgG secondary antibody (Rockland). Immunoreactive bands were visualized using an Odyssey Infrared Imaging System (Licor Biosciences, Lincoln, NE, United States).