Coomassie blue r 250 solution

Cells were lysed in a lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% sodium dodecyl sulfate, and 1X protease inhibitor cocktail). The protein amount per sample was quantified using the Pierce™ BCA protein assay kit (ThermoFisher Scientific, San Jose, CA, USA). Equal amounts of protein were loaded and resolved on 15% SDS-PAGE along with SeeBluePlus 2 pre-stained molecular weight standards (Life Technologies, Carlsbad, CA, USA). Proteins were prefixed in-gel with 50% methanol and 10% glacial acetic acid in water, then stained with Coomassie Blue R-250 solution (Sigma-Aldrich, St Louis, MO, USA) for 1 h and de-stained overnight with 40% methanol and 10% glacial acetic acid in water. An image of the gel was acquired on an Odyssey Fc imaging system using Image Studio software version 5.0 (Li-COR, Lincoln, NE, USA). Each gel lane was cut into 10 molecular weight fractions and proteins were in-gel digested with trypsin using an automated digestion procedure (Intavis AG, Koln, Germany) described elsewhere [30 (link)]. Eluates containing the peptides were dried by centrifugal vacuum concentration.

A gel zymography was performed in cell lysates after treatment of NPCs with TNF‐α and/or inhibition of the ERK signaling pathway to analyze the pattern of MMP2/MMP9 in different treatment groups. Briefly, the cell layers were washed with phosphate‐buffered saline (PBS) and homogenized with 0.5% Triton‐X (Sigma‐Aldrich) containing a protease inhibitor (Sigma‐Aldrich). The extracts were applied to a gelatin‐containing 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and the gel was incubated in a developing buffer composed of 50 mM Trizma Base and 5 mM CaCl₂ (pH 8.0) overnight at 37°C. Subsequently, the polyacrylamide gel was stained with 0.1% Coomassie blue R250 solution (Sigma‐Aldrich) for 2 h.