Bca pierce bca protein assay kit

One day after the last MnBuOE injection, a small cohort (n = 4 per) were sacrificed for immediate protein analysis. The hippocampus was removed and placed in 400 µl of RIPA lysis buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 M EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl). The tissue was homogenized on ice, incubated for 30 min on ice, and then centrifuged at 20,000× g for 10 min at 4 °C. The supernatant was transferred to a new microcentrifuge tube and stored at −80 °C until processing. The Compat-Able^TM Protein Assay Preparation Reagent Kit (Thermo Fisher Scientific Waltham, MA, USA) was used to eliminate EGTA as an interfering substance for the BCA Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein was separated by SDS-PAGE using the 4–15% Criterion™ TGX™ Precast Midi Protein Gel, 18-well, 30 µL (Bio-Rad, Hercules, CA, USA) ran at 120V for 75 min. The gel was stained using Coomassie Blue staining (Bio-Rad, Hercules, CA, USA). The samples were then sent to the UAMS Proteomics Core for further processing for mass spectrometry.

The hippocampus was removed and placed in 400 µL of RIPA lysis buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 M EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl). The tissue was homogenized on ice, incubated for 30 min on ice, and then centrifuged at 20,000 × g for 10 min at 4°C. The supernatant was transferred to a new microcentrifuge tube and stored at −80°C until processing. The Compat-Able™ Protein Assay Preparation Reagent Kit (Thermo Scientific™) was used to eliminate EGTA as an interfering substance for the BCA Pierce TM BCA Protein Assay Kit (Thermo Scientific™).

Cells cultured in T-25 flasks were washed three times with Dulbecco’s phosphate-buffered saline (Gibco 14190250) to completely remove culture media. Cells were lysed with cell lysis buffer (Sigma, C3228) supplemented with a protease inhibitor (Thermo Fisher Scientific, 78429). Cells were harvested by scraping and maintained on ice over a rocker for 30 min. Then, the samples were centrifuged at 10,000 g for 10 min at 4°C. The protein samples were then collected and quantified using the BCA pierce TM BCA Protein Assay kit (Thermo Fisher Scientific, 23225).