Journal: Scientific Reports
Article Title: Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics
Figure Lengend Snippet: Distinct HRNR-derived CIDAMPs show similar ribosomal protein binding patterns. ( a ) SulfoLink®–column-bound proteins of an E . coli -extract were separated on a Jupiter® C18 RP-HPLC column with a Prp-gradient. HPLC fractions containing UV-absorbing peaks (C1–D10) were divided into five aliquots and adjusted in parallel to five PAGE-gels and separated. ( b ) Silver-stained proteins. ( c ) HRNR-Far-Western blot for probing with biotinylated HR1-18 (HRNR 2556–2677 ) using Strep - Tactin ®, ( d ) HRNR-Far-Western blot for probing with biotinylated rSumo3-HRNR 2591–2684 using Strep - Tactin ®, ( e ) HRNR-Far-Western blot for probing with rHRNR 2591–2684 using anti-HRNR 2591–2684 antibodies, ( f ) HRNR-Far-Western blot for probing with rHRNR 1075–1172 using anti-HRNR 1075–1172 antibodies. Note similarities of the staining patterns, irrespective the CIDAMP AA-sequence or biotin-labeling and irrespective whether a Strep - Tactin ®- or antibody-detectable CIDAMP was used to probe and detect the target protein on the membrane. Note the presence of 70 kDa bands upon HRNR-Far-Western blot analyses in most of the investigated HPLC fractions with highest intensity for rHRNR 1075–1172 binding ( f ). The most intensive band, corresponding to a 37 kDa protein in fraction number C13, was identified as E . coli ribosomal protein L2.
Article Snippet: We either used a Jupiter® 300 Å, 250 × 12.6 mm, C18-RP-HPLC column (Phenomenex) or an Aeris® widepore XB C18, 250 × 12.6 mm, RP-HPLC column (Phenomenex) and either a gradient of acetonitrile (ACN) in aqueous 0.1% TFA, or a gradient of 2-propanol (Prp) in aqueous 0.1% TFA – as indicated for separation of ribosomal proteins.
Techniques: Derivative Assay, Protein Binding, High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis, Staining, Far Western Blot, Sequencing, Labeling, Binding Assay