Gel and Blot Imaging

AlkPhos Direct™ Labelling and Detection Systems provide a non-radioactive alternative for use in Southern and Northern blot hybridizations. By covalently cross-linking the nucleic acid probe to alkaline phosphatase, the system circumvents the need for extensive antibody incubations/washings that are typical in hapten-based systems. With fewer steps, less time is required, and user error is less likely. Blots are typically developed using the chemiluminescent substrate CDP-Star™, after which the blots are exposed to film. However, more investigators with access to fluorescence imaging systems are now switching to chemifluorescent detection because it offers a greater range of linearity in quantification. This report compares the development of duplicate Southern blots using the chemifluorescent substrates ECF™ detection reagent and DDAO phosphate.

Rosemary (Rosmarinus officinalis) genomic DNA (500 ng, 250 ng, 125 ng, and
62.5 ng) was digested with Hind III and loaded in duplicate onto a 0.8% agarose gel. Following electrophoresis, the DNA bands were transferred onto a Hybond™- N+ nylon membrane by vacuum blotting. The membrane was cut in half, and the two blots were pre-hybridized in AlkPhos Direct hybridization buffer for 1 h at 55 °C. An 800 bp ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) probe was labelled with AlkPhos Direct for 2 h at 50 °C, rather than the standard 30 min at 37 °C because greater sensitivity with chemifluorescent detection could be achieved with these labelling conditions. The probe was added to the hybridization buffer at a concentration of 10 ng/ml and incubated with the blot overnight at 55 °C.

Following hybridzation, the blots were washed twice for 10 min at 55 °C using primary wash buffer, and twice for 5 min at room temperature in secondary wash buffer. The blots were then transferred into separate detection bags and developed using either the ECF substrate supplied in the ECF Detection Module or a 1:1000 dilution of DDAO phosphate stock solution (1.25 mg/ml) in 10 mM Tris (pH 9.5) containing 1 mM MgCl₂. The blots were scanned at intervals of 30 min, 1 h, 2 h, 6 h, and 24 h on Storm™ 860 variable mode imager using either the blue fluorescence mode for ECF (Ex_max 440 nm) or the red fluorescence mode for DDAO phosphate (Ex_max 646 nm). All scans were performed using a PMT setting of 900 V.

As seen in Figure 1a, although DDAO phosphate develops quickly, background and band diffusion increase significantly with extended incubation. In contrast, ECF develops much more slowly than DDAO phosphate, with little band diffusion and almost no increase in background during longer incubations (Fig 1b).

(a) DDAO phosphate



(b) ECF



Fig 1. Duplicate Southern blots were developed with either (a) DDAO phosphate or (b) ECF and scanned at the time intervals indicated (see main text for details). All images were normalized over the full range from 1–100 000 rfu to facilitate direct comparison and consequently may not be the optimal image for each individual blot.

While both substrates can be used for alkaline phosphatase-based chemifluorescent detection, the choice of substrate will depend on the scanner available. Storm 830 has a red fluorescence mode, but no blue fluorescence mode, so DDAO phosphate would be required for detection. Conversely, Storm 840 has a blue fluorescence mode, but no red fluorescence mode, making ECF the substrate of choice. Storm 860 has both blue and red fluorescence modes, so substrate choice would be at the discretion of the user.