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Co-expression with the Type 3 Secretion Chaperone CesT from Enterohemorrhagic E. coli Increases Accumulation of Recombinant Tir in Plant Chloroplasts

Frontiers in Plant Science. 2017;

Jacqueline MacDonald , Sean Miletic,† , Typhanie Gaildry, , Adam Chin-Fatt, and Rima Menassa *

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Peptide Synthesis	Liquid protein samples were combined with 5x loading buffer (0.3 M Tris-HCl pH 8.0, 5% SDS, 10% glycerol, 100 mM DTT, 0.05% Phenol Red), heated at 99◦C for 5 min, then loaded onto Express Plus PAGE 4-20% gels (Genscript Inc., Piscataway, NJ, USA). Gels were run at 100 V for 100–110 min, then transferred to polyvinylidine difluoride (PVDF) membrane using the TransBlot Turbo transfer system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Blots were blocked with 5% skimmed milk in trisbuffered saline, pH 7.5, and proteins of interest were probed with anti-c-myc antibody from mouse (Genscript Inc., Piscataway, NJ, USA) and the One-Hour Basic Western kit for mouse primary antibody (Genscript Inc., Piscataway, NJ, USA).Protein sizes were predicted based on amino acid sequence only, using an online peptide calculator tool (Genscript Inc.; https://www.genscript.com/sslbin/peptide_mw).	Get A Quote