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Detection Methods

In addition to selecting the optimal imager or scanner for the job at hand, the most appropriate choice of label or stain will also enhance performance in terms of both sensitivity and dynamic range.

In 1975, the British biologist, Edwin Southern, pioneered the use of isotope-labeled nucleotides for detection of specific sequences in DNA. Today, radioisotopes have lost their position as the principle detection technology to safer, cheaper, and more quantitatively precise light-emitting technologies based on chemiluminescence and fluorescence.

Chemiluminescence Fluorescence Chemifluorescence Other Methods

Chemiluminescence occurs when a chemical reagent containing stored energy releases light. The reagent is normally stable and does not emit light, but can be converted into a light emitting product, for example after interaction with a specific enzyme. In most contemporary ECL systems, the enzyme horseradish peroxidase (HRP) conjugated to a secondary antibody is utilized to convert the substrate, producing light.

Fluorescence occurs when molecules called fluorophores absorb light. In their ground state, fluorophores do not emit light, but when subjected to light (excitation) their energy levels are raised to a brief but unstable excited state. As fluorophores return to their ground state, they release light at a lower energy, higher wavelength (emission) than that of the excitation light. If selected fluorescent dyes are spectrally resolvable (i.e., emit light of different wavelengths), they can be used as labels to allow multiplexing – the simultaneous detection of more than one target in a single sample.

Chemifluorescence refers to the chemical and/or enzymatic production of fluorescence. A catalytic reaction between enzyme and substrate results in the formation of a fluorescent product at the site of the reaction as well as an amplification of the signal.

Colorimetric Methods

Colorimetric Methods using dyes such as Coomassie Blue and silver staining are still widely used to visualize and detect proteins. Silver can be used to detect as little as sub-nanogram quantities of protein.

Radioisotopes

Although alternatives to radioisotope-based detection systems have emerged, there are still some areas in which radioisotopes such as 32P, 33P, 35S, 14C, 3H and 125I continue to offer some advantages. Radioactive systems in combination with phosphorimaging screens are still used in Southern and Northern blotting because of their sensitivity and speed.