Nap 5 sephadex g 25 dna grade columns

For further characterization, mAb14 and Nimotuzumab stressed samples were treated as follows. 250 µg of mAb was denatured by addition of denaturing buffer (0.4 M Tris (Sigma-Aldrich, Taufkirchen, Germany), 8 M guanidinium hydrochloride (Sigma-Aldrich, Taufkirchen, Germany), pH 8) to a final volume of 240 µL. Reduction was achieved by addition of 20 µL of 0.24 M dithiothreitol (DTT) (Roche, Mannheim, Germany) freshly prepared in denaturing buffer and incubation at 37 °C for 60 min. Subsequently, the sample was alkylated by addition of 20 µL of 0.6 M iodoacetic acid (Merck, Darmstadt, Germany) in water for 15 min at room temperature in the dark. The excess of alkylation reagent was inactivated by addition of 30 µL of DTT solution. The samples were then buffer exchanged to approximately 480 µL of 50 mM Tris/HCl, pH 7.5 using NAP5 Sephadex G-25 DNA grade columns (GE Healthcare, Germany). The mAbs were digested 5 h at 37 °C by addition of 0.03 µg trypsin (Promega, Madison) per µg protein in a final volume of 500 µL. Digestion was stopped by addition of 20 µL of 10% formic acid (FA)-solution, and samples were frozen at −80°C until further analysis.