Mouse anti his monoclonal antibody

Fifteen-day-old hypocotyls were fixed overnight at 4 °C in 4% paraformaldehyde (Sigma-Aldrich, Steinheim, Germany) in phosphate buffered saline (PBS). After washing, sections of 5 mm were embedded in 5% agarose. Cross sections of 100 µM were cut using a vibratome (Leica Biosystems, Nussloch, Germany) and were stained with toluidine blue (Sigma-Aldrich), or used for cellulose immunodetection. The cellulose-binding module CBM3a probe (PlantProbes, Leeds, UK) specific for crystalline cellulose was diluted to 10 µg/mL in milk protein/PBS (MP/PBS, 5% w/v), incubated in mouse anti-His monoclonal antibody (1% in MP/PBS, Sigma-Aldrich) and finally incubated in 2% anti-mouse IgG coupled to fluorescein isothiocyanate (FITC) (Sigma-Aldrich) in MP/PBS. CBM3a incubation lasted for 1.5 h. Anti-His and IgG-FITC incubation lasted 1 h. Between each step, three washes of 5 min each with PBS were performed. The slides were mounted in 50% glycerol (Sigma-Aldrich) and observed with a confocal microscope LSM 510 Meta (Zeiss, Jena, Germany) with the following setting: excitation at 488 nm, filter HFT 488/594 and emission recorded with LP 505, as previously reported [19 (link)].

The bacteria including Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibrio anguillarum, Vibrio parahemolyticus, Acinetobacter calcoaceticus and Saccharomyces cerevisiae were inoculated and cultured as described (6 (link)). The cells were washed and resuspended in PBS buffer. Approximately 2×10⁶ microbes were incubated with 1 μg of purified recombinant proteins in PBS by gentle orbital rotation overnight at 4°C. Microbes were pelleted and washed five times with 1 ml of PBST (0.05%Tween-20 in PBS). The washed pellets were then suspended with reducing sample buffer quickly and denatured by heating at 100°C for 10 min. The binding proteins were validated by Western blot with an anti-His mouse monoclonal antibody (Sigma).

Bacteria Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibrio anguillarum, Vibrio parahemolyticus, Acinetobacter calcoaceticus and Saccharomyces cerevisiae were respectively inoculated into suitable liquid medium and cultivated overnight under suitable conditions. V. anguillarum was cultured with high-salt Luria-Bertani (LB) medium at 28°C, V. parahemolyticus was cultured with seawater medium at 28°C, S. cerevisiae was cultured with YPD medium at 28°C, and the remaining bacteria were cultured with LB medium at 37°C. The next day, the cells were collected and washed with PBS buffer and resuspended in PBS. Approximately 2×10⁶ microbes were incubated with 1 μg of purified recombinant proteins in 1 mL PBS at 4°C overnight with gentle orbital rotation. Microbes were centrifuged at 12,000 × g for 1 min at 4°C and the pellets were washed five times with 1 mL of PBST (0.05% Tween-20 in PBS). The washed pellets were then suspended in 100μl PBS and 20μl 6×loading buffer and boiled at 100°C for 10 min. Western blot was performed with an anti-His mouse monoclonal antibody (Sigma) to validate the binding proteins.

A biotinylated double-stranded DNA probe was formed by heating 400 pmol biotinylated oligonucleotide pairs, shown in Supplementary Data 6, to 95 °C. Separately, 3 μg purified CnHsf3-part1 protein was added to a 50-fold molar excess of NaClO and held for 60 min at room temperature. The protein expression and purification procedures are provided in the Supplementary Methods. The biotinylated double-stranded DNA probe was incubated with 40 μl Streptavidin Magnetic Beads (MedChemExpress) and mixed with the protein preparation. The mixture was incubated on a rocking platform at room temperature for 2 h with gentle agitation. The beads were washed three times with Buffer I (10 mM Tris–HCl [pH 7.5], 1 mM EDTA, 1 M NaCl, and 0.05% Tween-20), then resuspend in 40 μl protein loading buffer and incubated for 5 min at 95 °C. Protein samples were separated using 12% SDS–PAGE electrophoresis, transferred onto a nitrocellulose membrane, and blocked with 5% milk. Immunoblotting assays were performed using anti-His mouse monoclonal antibody (1:5000 dilution; Sigma) and goat anti-mouse IgG (H + L) HRP secondary antibody (1:5000 dilution; Thermo Fisher Scientific) antibodies, and the signal was captured using a ChemiDoc XRS + (Bio-Rad). The Electrophoretic mobility shift assay (EMSA) was performed as described in the Supplementary Methods.