Recombinant glucagon peptide expression profiling and analysis. (A) Glucagon expression after different incubation times. Upper lane: protein expression using 1 L of culture media; Lower lane: protein expression in the bioreactor (5 L). Lane 1: BenchMark Protein Ladder (Invitrogen); Lanes 2 to 9 indicate expression protein patterns after 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and overnight incubation, respectively. (B) Western blotting of glucagon using anti-glucagon antibodies. Lane 1: Pre-Stained Protein Ladder (Invitrogen); Lane 2: purified GST protein (3 μg); Lane 3 purified non-digested glucagon fused with the GST protein (3 μg) arrows indicate recombinant glucagon fused with GST protein. (C) Glucagon digestion to separate the peptide from the GST tag in Tris-Tricine gel. Lane 1: Seeblue Plus2 Pre Stained Standard (Invitrogen); Lane 2: purified non-digested glucagon fused with the GST protein (5 μg); Lane 3: Pre-Stained Protein Ladder (Invitrogen); Lane 4: commercial glucagon (Glucagen, NovoNordisk, 5 μg); Lane 5: digested glucagon (40 μg total digestion).