Sensitivity: 0.03 pg of homologous DNA or 0.1 pg of homologous RNA is detected in a Southern or dot blot after <30-min exposure time. Single-copy genes are detected in 0.3 µg mammalian genomic DNA.

Figure 1: Detection of DIG-labeled nucleic acids with CSPD. Human genomic DNA was digested with Eco RI, separated on a 1% agarose gel, and blotted onto positively charged nylon membranes. The blots were hybridized with 50 ng/ml DIG-labeled β-actin RNA. Chemiluminescent detection was performed according to the standard DIG chemiluminescent-detection procedure, using CSPD at a 0.25-mM final concentration.

Background Information

Principle

• DIG-labeled nucleic-acid probes are used for hybridization in various blot applications.
• After hybridization, the blots are submitted to immunological detection using a highly specific anti-digoxigenin-antibody, conjugated to alkaline phosphatase.
• The addition of the chemiluminescence substrate CSPD leads to a distinct luminescent signal that is recorded on X-ray or Polaroid film.
• Exposure time varies between 5 and 20 min, and because of the long-lasting glow, multiple exposures can be taken over several days.
• Blots developed with CSPD can be easily stripped and reprobed.

Figure 2: CSPD reaction.