Coomassie brilliant blue stain

The conjugated probes were purified from unconjugated antibody and oligonucleotide by ÄKTA Pure chromatography (GE Healthcare) using a Superdex 200 10/300 GL column (GE Healthcare). Successful purification was confirmed by separating the conjugates on a Novex TBU 10% gel (Life Technologies) at 150 V for 60 min in a water bath pre-heated to 50 °C. DNA was visualized using SYBR Gold Nucleic Acid Gel Stain (Life Technologies), and protein was visualized using Coomassie brilliant blue stain (Bio-Rad). The gel was imaged on Odyssey Fc with the Image Studio Lite v5.2.5 software (LI-COR Biosciences).

Crude extracts were mixed with 6x SDS sample application buffer [25% (v/v) glycerol, 0.5 M DDT, 5% (w/v) bromothymol blue] to a final concentration of 1x and boiled at 90°C for 10 min. Fifteen per cent SDS-PAGE-gels were loaded and electrophoresed in a Bio-Rad Tetra Cell system at 120 volts. Gels were stained with Aqua Stain^™ (Vacutec) for 1 h, at 37°C, or with Coomassie Brilliant Blue stain (Bio-Rad) for 2 h at 37°C and destained with destaining solution [30% (v/v) methanol and 10% (v/v) glacial acetic acid in distilled water. Protein sizes were estimated using PageRuler^™ Plus Prestained Protein Ladder (Thermo Scientific) or with the Colour Prestained Protein Standard, Broad Range (11–245 kDa) (NEB).
For western blots, proteins were transferred from SDS-PAGE-gels to nitrocellulose membranes using a Bio-Rad Trans-Blot^® Semi-dry transfer cell at 15 volts, for 1 h. The primary and secondary antibodies used were the polyclonal anti-HPV-16-E7 rabbit serum (I. Hitzeroth, Biopharming Research Unit, MCB, UCT) and the goat anti-Rabbit IgG whole molecules-AP (Sigma-Aldrich) unless otherwise stated. Blots were visualized with NBT/BCIP substrate (Sigma-Aldrich).