Criterion stain free gel doc ez imager

The molecular weight distribution of control and hydrolysed samples was determined according to Laemmli [40] using SDS-PAGE under reducing conditions. The samples were diluted with Tris-HCl treatment buffer (0.125 mol L -1 Tris-HCl, 4% SDS, 20% v/v Glycerol, 0.2 mol L -1 DTT, 0.02% bromophenol blue, pH 6.8). Samples were boiled for 3 min to cleave noncovalent bonds and centrifuged at 14,000×g for 10 min (Mini Spin, Eppendorf AG, Hamburg, Germany). The electrophoresis was performed on 4-20% midi Criterion™ TGX Stain-Free™ precast gels (Bio-Rad, Ismaning, Germany) and the proteins were separated using the Midi Criterion™ Cell (Bio-Rad, Ismaning, Germany) and an electrophoretic power supply (E835, Consort, Turnhout, Belgium). A molecular weight marker containing recombinant protein bands in the range of 10-250 kDa (Precision Plus Protein™ Unstained Standard, Bio-Rad Laboratories Inc., Hercules, CA, USA) was additionally loaded onto the gel. Electrophoresis conditions were 200V, 60 mA, 100 W at room temperature. Protein visualisation and evaluation were performed by Criterion Stain-Free Gel Doc™ EZ Imager (Bio-Rad, Ismaning, Germany).