Journal: bioRxiv

Article Title: Evolutionary engineering a larger porin using a loop-to-hairpin mechanism

doi: 10.1101/2023.06.14.544993

Figure Lengend Snippet: (A, B) Western blots with the nitrocellulose membrane stained with Ponceau S and subsequently immunoblotted against various antibodies. (A) Western blot of different fractions of E. coli displaying the localization of the his-tagged recombinant proteins PorB WT and PorB 18 in the outer membrane. SurA, a periplasmic protein, indicates the localization of periplasmic proteins. Whole-cell fractions (WC), soluble (S), inner membrane and trapped periplasmic protein (IM/P), and outer membrane (OM) (B) Western blot displaying the susceptibility of proteins to Proteinase K under different conditions. His-tagged PorB WT (blue asterisks) and PorB 18 (green asterisks) show resistance to Proteinase K when not boiled but are partially susceptible when heat denatured. TolC (red asterisks) is used as a positive control as it shows a 5 kDa cleavage in the presence of proteinase K resulting in a 45 kDa cleavage product (red arrows) .

Article Snippet: The following antibodies were diluted as per manufacturer recommendation with TBST with 1% gelatin: THE Anti-His tag monoclonal mouse antibody (GenScript), anti- E. coli SurA polyclonal rabbit antibody (Cusabio), anti- E. coli BamA polyclonal rabbit antibody (Cusabio), anti- E. coli GroEL polyclonal rabbit antibody (Enzo Life Sciences), IRDye 800CW goat anti-mouse IgG secondary antibody (LI-COR Biosciences), IRDye 680RD donkey anti-rabbit IgG secondary antibody (LI-COR Biosciences). anti- E. coli TolC was purified from rabbit sera and diluted to 1:2500 with 1% gelatin in TBST.

Techniques: Western Blot, Membrane, Staining, Recombinant, Positive Control

Journal: bioRxiv

Article Title: Evolutionary engineering a larger porin using a loop-to-hairpin mechanism

doi: 10.1101/2023.06.14.544993

Figure Lengend Snippet: (A) Single channel conductance of PorB 18 (blue) compared to PorB WT (green). (B) Box and whisker plot comparing the conductance of PorB WT (green) and PorB 18 (blue). ****: p-value <= 10 -4 (C) Graph shows comparison of experimental single-channel conductance of PorB WT with His-Tag (green) and without His-tag (light green), PorB 18 (blue), theoretical shingle-channel conductance of AlphaFold 18-stranded model (magenta) and 16-stranded model (cyan) of PorB 18 . (D) Channel conductance at 100 mV showing channel closing for individual subunits of PorB WT (green) and PorB 18 (blue). Shortened loop constructs collapse

Article Snippet: The following antibodies were diluted as per manufacturer recommendation with TBST with 1% gelatin: THE Anti-His tag monoclonal mouse antibody (GenScript), anti- E. coli SurA polyclonal rabbit antibody (Cusabio), anti- E. coli BamA polyclonal rabbit antibody (Cusabio), anti- E. coli GroEL polyclonal rabbit antibody (Enzo Life Sciences), IRDye 800CW goat anti-mouse IgG secondary antibody (LI-COR Biosciences), IRDye 680RD donkey anti-rabbit IgG secondary antibody (LI-COR Biosciences). anti- E. coli TolC was purified from rabbit sera and diluted to 1:2500 with 1% gelatin in TBST.

Techniques: Whisker Assay, Comparison, Construct

Journal: Frontiers in Microbiology

Article Title: Novel anti-CEA affibody for rapid tumor-targeting and molecular imaging diagnosis in mice bearing gastrointestinal cancer cell lines

doi: 10.3389/fmicb.2024.1464088

Figure Lengend Snippet: Binding of Z CEA affibodies to HT-29 and MKN-45 cells by indirect immunofluorescence assay. (A) HT-29 (B) MKN-45 cells were incubated with Z CEA affibodies. Mouse-anti-His-tag mAb FITC (green) was used to visualize the binding of Z CEA affibodies to CEA+ cancer cell lines and nuclei were counterstained with Hoechst 3342 (400×). (C) HeLa229 served as the CEA cancer cell line. Z WT set as the affibody negative control. Scale bar = 10 μm. Experiments were performed in triplicate.

Article Snippet: The molecular weight and purity of the fusion proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using an anti-His-tag monoclonal antibody (MultiSciences Biotech Co., Ltd., Hangzhou, China).

Techniques: Binding Assay, Immunofluorescence, Incubation, Negative Control