Imagequant las 4000 mini camera system

Leaf genomic DNA was extracted from young leaves (2 g) as above. Standard procedures (Sambrook et al., 1989 ) were used. Genomic DNA (~20 µg) was digested with EcoRI or SacI, followed by fractionation on a 0.7% (w/v) agarose gel and blotting onto a Hybond N+ nylon membrane (GE Healthcare). The CTB DNA probe was amplified from pZAAMP-CTB-10Li45GB3A by PCR with the CTB-F and -R primers (Supplementary Table 2). An AlkPhos Direct Labelling Module and Detection System with CDP-Star Detection Reagent (GE Healthcare) were used. Bands were detected with an ImageQuant LAS 4000 mini camera system (GE Healthcare). pZAAMP-CTB-10Li45GB3A was used as a positive control, and WT rice genomic DNA was used as a negative control.

The CRM₁₉₇ protein or MenX-CRM, MenA-CRM₁₉₇, and GBSII-CRM₁₉₇ glycoconjugates in the amount of 2–10 μg were separated by 8% SDS-PAGE. Fab fragments in the amount of 2–10 μg were separated by 10–12% SDS-PAGE. Samples were transferred onto a 0.45 μm PVDF membrane (Hybond™, GE Healthcare), which were subsequently blocked with 5% w/v blotting grade low-fat powdered milk (Carl Roth Gmbh & Co. Kg). Membranes were incubated with clone MenX.01 (mAb or Fab) overnight at 4 °C. We used our own stock antibodies at a concentration of 1 mg/ml with a typical dilution of the primary antibody being 1:100. Protein signals were developed using anti-mouse IgG F (ab’) 2 peroxidase (Jackson ImmunoResearch) 1:1,000 and visualized with an ImageQuant LAS 4000 mini camera system (GE Healthcare). Fab fragments were developed with either anti-mouse IgG F (ab’)2 peroxidase (Jackson ImmunoResearch) diluted 1:1,000 or anti-mouse IgG (H + L) Fc peroxidase (Jackson ImmunoResearch) diluted 1:1,000 to confirm the absence of the Fc fragment in the preparation.