Bio safe coomassie brilliant blue g 250

Measurements were performed using six different inhibitor concentrations starting at 200 μM of 11 with three-fold dilution. Human recombinant, His6-SENP1 Catalytic Domain (Boston Biochem Cat# E-700, Bio-Techne AG, Zug, Switzerland) was prepared in reaction buffer (50 mM HEPES, 150 mM NaCl 0.5 mM EDTA, 1 mM DTT, pH 7.5, 0.01% CHAPS) as 555 nM solution. A 20 mM compound DMSO stock-solution (1.1 µL) was added to the freshly prepared 555 nM enzyme solution (98.9 µL). Five sequential three-fold dilutions resulted in a final inhibitor concentration of 0.8 µM and the enzyme inhibitor complex was incubated for 15 min at 0 °C. Then, 50 µM proSUMO3 (5 µL) was added to the freshly prepared enzyme inhibitor solution (45 µL) and incubated for 60 min at 37 °C and subsequently the reaction was stopped by adding 4 × Laemmli-Buffer (16.7 µL) and denaturing the protein at 100 °C for 30 min. Those solutions (10 µL) were loaded on a casted 15% sodium dodecyl sulfate polyacrylamide gel and separated on a Mini-PROTEAN Tetra Cell (Bio-Rad, Hercules, CA, USA). After SDS-PAGE gel electrophoresis separation, the gels were stained with staining solution Bio-Safe Coomassie Brilliant Blue G-250 (BioRad) for 60 min. De-staining was performed with 40% (v/v) methanol, 10% (v/v) glacial acetic acid, 50% (v/v) Milli-Q water until protein bands became visible.

Protein concentrations were measured by Bradford assay (Bradford, 1976 (link)) using bovine serum albumin (BSA, Bradford reagent 5 ×, Bio-Rad, Hercules, CA, United States) as standard. The proteins from supernatant and cell lysate samples were concentrated and exchanged to 0.1 M sodium phosphate buffer pH 6.5 using Amicon Ultra 0.5 mL Centrifugal filters (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) with 10 kDa cut-off prior to SDS-PAGE. The protein bands were visualized using Bio-Safe Coomassie Brilliant Blue G-250 (Bio-Rad, Hercules, CA, United States).
α-Amylase activity was measured using the DNS method (3,5-dinitrosalicylic acid, Sigma-Aldrich, St. Louis, MI, United States) as described previously (Kanpiengjai et al., 2015a (link)) using D-glucose as standard. One unit of amylase activity was defined as the amount of enzyme releasing 1 μmol of reducing sugars (or reducing end equivalents) per minute under the given conditions.