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. 2021 Apr 15;9:e11063. doi: 10.7717/peerj.11063

Figure 7. SDS-PAGE analysis of recombinant immunotoxins.

Figure 7

(A) Protein expression was induced with 1 mM IPTG for 3 h at 30 °C. The samples were collected before and after induction. The cell pellets were resuspended in 2X Laemmli sample buffer and heated to denature. After centrifugation at 10,000g for 2 min, the supernatant was loaded on 12% (w/v) SDS- PAGE and stained with Coomassie brilliant blue. The IPTG-induced scFv-mHALT-1 (Lane 2) and mHALT-1 scFv band (Lane 4) at 44.4 kDa and 46.8 kDa, respectively, are shown with arrows. Uninduced scFv-mHALT-1 and mHALT-1-scFv were loaded on Lane 1 and Lane 3, respectively. (B) Denatured recombinant immunotoxins were refolded by step-wise dialysis using Snakeskin dialysis tubing (MWCO: 10 K) with decreasing concentrations of urea (6 M, 4 M, 2 M, 1 M) and final dialysis in PBS containing 25% glycerol. The refolded recombinant immunotoxins scFv-mHALT-1 (Lane 1) and mHALT-1-scFv (Lane 2) were loaded on 12% SDS-PAGE, and proteins were stained with Coomassie Brilliant Blue. Lane M shows the pre-stained broad range protein marker (NEB, Hitchin, UK).