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: Scientific Reports

Article Title: His-tag protein monitoring by a fast mix-and-measure immunoassay

doi: 10.1038/srep05613

Figure Lengend Snippet: The donor-labeled peptide binds to the acceptor-labeled antibody. The donor emission is reduced due to FRET. The His-tag-containing analyte in the sample displaces the donor peptide, which leads to a concentration-dependent signal increase. EuLH was used as donor dye for phosphorescence detection, BHQ-10 served as an acceptor dye on the antibody.

Article Snippet: The monoclonal mouse anti-His-tag antibody “8-4-4” was generated in collaboration with Biogenes (Berlin, Germany).

Techniques: Labeling, Concentration Assay

Journal: Scientific Reports

Article Title: His-tag protein monitoring by a fast mix-and-measure immunoassay

doi: 10.1038/srep05613

Figure Lengend Snippet: Each donor peptide (10 μL, 40 nmol/L) was mixed with increasing concentrations of BHQ-10-labeled anti-His-tag mAb “8-4-4” (40 μL, 0…160 nmol/L). Fluorescence intensity was measured after a 90 s shaking step. Error bars = SD, n = 3.

Article Snippet: The monoclonal mouse anti-His-tag antibody “8-4-4” was generated in collaboration with Biogenes (Berlin, Germany).

Techniques: Labeling, Fluorescence

Journal: Scientific Reports

Article Title: His-tag protein monitoring by a fast mix-and-measure immunoassay

doi: 10.1038/srep05613

Figure Lengend Snippet: Homogeneous His 6 detection assay – Response curves for C-terminally His 6 -tagged TEV protease (a), N-terminally His 6 -tagged trigger factor (b) and chemically labeled (His 6 ) chem -BSA/tag-free BSA as a control (c). Protein samples (50 μL) were premixed with donor peptide (10 μL, 40 nmol/L) and then mixed with BHQ-10-labeled anti-His-tag mAb “8-4-4” (40 μL, 80 nmol/L). Phosphorescence intensity was measured after a 90 s shaking step. Error bars = SD, n = 3. The grey dashed lines indicate the detection limit (Background signal c = 0 plus 3x SD).

Article Snippet: The monoclonal mouse anti-His-tag antibody “8-4-4” was generated in collaboration with Biogenes (Berlin, Germany).

Techniques: Detection Assay, Labeling, Control

Journal: Scientific Reports

Article Title: His-tag protein monitoring by a fast mix-and-measure immunoassay

doi: 10.1038/srep05613

Figure Lengend Snippet: His 6 samples (TEV-His 6 , 50 μL, dissolved in E. coli cell lysate or buffer) were premixed with donor peptide (10 μL, 40 nmol/L) and then mixed with BHQ-10-labeled anti-His-tag mAb “8-4-4” (40 μL, 80 nmol/L). Phosphorescence intensity was measured after a 90 s shaking step. Error bars = SD, n = 3.

Article Snippet: The monoclonal mouse anti-His-tag antibody “8-4-4” was generated in collaboration with Biogenes (Berlin, Germany).

Techniques: Labeling

Journal: Scientific Reports

Article Title: His-tag protein monitoring by a fast mix-and-measure immunoassay

doi: 10.1038/srep05613

Figure Lengend Snippet: Standard: Precision Plus Protein ™ Dual Xtra (Bio-Rad Laboratories); Lane 1: (His 6 ) chem -BSA (1 μg); lane 2: TEV-His 6 in buffer (1 μg); lane 3: TEV-His 6 (1 μg) in E. coli cell lysate. RPE-goat-anti-mouse-IgG was used as secondary antibody for fluorescence detection (605/50 nm, recording time 5.33 s).

Article Snippet: The monoclonal mouse anti-His-tag antibody “8-4-4” was generated in collaboration with Biogenes (Berlin, Germany).

Techniques: Fluorescence