α chymotrypsin

In-gel digestion of NTL-125 was performed to determine the protein sequence. The purified protein band was excised from the gel. The CBB stain was removed from gel piece by incubating with 50% 25 mM NH₄HCO₃ and 50% (v/v) acetonitrile solution. The destained gel pieces were dehydrated with 100 % acetonitrile (ACN) for 10 min in a microcentrifuge tube followed by drying in a vacuum centrifuge. Dried gel pieces were further reduced with 10 mM DTT for 45 mins at 55°C and alkylated with 55 mm iodoacetamide for 30 mins at room temperature in dark. After removing the iodoacetamide the gel pieces were washed with 50% 25 mM NH₄HCO₃ and 50% (v/v) acetonitrile solution at room temperature. The protein in the gel pieces was then cleaved with Trypsin (Gold Trypsin, Promega, USA) and α-Chymotrypsin (SRL, India) individually using 1:20 ratio of enzyme to substrate in 25 mM ammonium bicarbonate buffer (pH 8.0). The gel pieces with enzyme solutions were incubated on ice for 1hr. Thereafter sufficient amount of 25 mM NH₄HCO₃ was added to dip the gel pieces and incubated at 37°C overnight (Gundry et al., 2009 ). Next day the supernatant containing the peptides was collected in a new microcentrifuge tube. Digested peptides were further extracted from the gel pieces with 50% (v/v) acetonitrile, 1% (v/v) formic acid and 49% H₂O and finally dried using vacuum centrifuge.