Nupage 4 12 bt gel

Chromogenic substrate pGlu-Phe-Lys-pNA (S-2403) was from Chromogenix (Sweden). 4-Nitrophenyl 4-guanidinobenzoate hydrochloride (pNPGB) was from Aldrich. NUPAGE 4–12% BT GEL was from Invitrogen. Other chemicals and protein reagents were from SIGMA/Aldrich. Kinetic measurement was performed similarly as described^{47 (link)}. Briefly, the refolded and purified μPlg zymogens (35.5 µM) were activated with a plasminogen activator such as urokinase (20:1) at 37 °C for 4 min in a reaction mixture containing 25 mM Tris–HCl, pH 7.4, 50 mM NaCl. The active site of the activated μPlm was titrated using pNPGB as described^{52 (link)}. The activated zymogens were diluted to 5.5 µM, and then 10 µl was mixed with 100 µl of 0.0625 mM, 0.125 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, 1.5 mM, or 2.0 mM of substrate S-2403 in the assay buffer (25 mM Tris–HCl, 50 mM NaCl, pH 7.4). The generation of amidolytic activity was monitored (at 405 nm) at 37 °C in 10 s intervals for 20 min using SpectraMax 250 microplate reader (Molecular Devices). The data was plotted as velocity vs. substrate using GraFit version 7 (Erithacus Software) and the V_max and K_m of the wild-type and each mutant µPlm were determined. The catalytic efficiency (Kcat/Km) was calculated according to the active enzyme concentration.

Cortices and hippocampi of mice of indicated ages were lysed in ice-old RAPI buffer containing protease inhibitors (Roche). Proteins were electrophoresed on NUPAGE 4–12 % BT Gel (Invitrogen) and transferred to nitrocellulose membranes. Membranes were blocked with 5 % non-fat milk in PBS buffer for 1 h, incubated with primary antibodies at 4 °C overnight and with secondary antibodies for 1 h at room temperature after washing 3 times with PBST.