Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A i) SDS-PAGE analysis showing the purity of the Ni-NTA purified Z EGFR 1907’ under pseudo native and denatured conditions. Pseudo native gel bands (boxed) were extracted and analysed by mass spectrometry. Mass spectrometry data confirmed that both bands correspond to one species, (A ii) DNA sequencing of the plasmid revealed the above sequence and this was also confirmed by mass spectrometry analysis (B i) Elution profile of the size exclusion gel filtration chromatography of Z EGFR 1907’ affibody purified via His-Tag Ni-NTA purification. Superdex 75 1660 column equilibrated with 20 mM Hepes pH 7.4, 500 mM NaCl, 10% Glycerol and 2 mM β mercaptoethanol and 1 ml fractions were collected. Insert shows the complete elution profile. (B ii) 20 µl of selected sample fractions from the purification were denatured, run on an SDS-PAGE gel and stained with Coomassie blue. (C) Silver stained SDS-PAGE gel of the concentrated 1:1 triconjugate Z EGFR 1907’ (PPEA) under pseudo native and denatured conditions. Pseudo native and denatured conditions are indicated by -/+ β-mercaptoethanol.

Article Snippet: The final 1:1 Triconjugate Affibody-polyIC-polyplex (Z EGFR 1907’- polyIC-polyplex) was determined to be 100 μg/ml using the copper assay [ ].

Techniques: SDS Page, Purification, Mass Spectrometry, DNA Sequencing, Plasmid Preparation, Sequencing, Filtration, Chromatography, Staining