Detection of Proteins and Nucleic Acids

A great many techniques have been developed for the detection of proteins and nucleic acids. Previously, we discussed chromatography and spectrophotometry. Both of these techniques can be used to detect proteins and nucleic acids. For instance, both can be detected by their absorption of a specific wavelength of UV light. The peak absorption of proteins occurs at 280 nm, whereas the peak absorption of nucleic acids occurs at 260 nm.

2.6.1.1 Principles of Electrophoresis

Probably the most common technique employed in the laboratory to detect proteins or nucleic acids is electrophoresis. Electrophoresis is a technique by which the movement of charged molecules in an electric field can be studied. Most people have likely heard of the term "gel electrophoresis," which employs a buffer-saturated gel matrix as a support medium to which the sample to be analyzed is applied. The migration of the sample through the matrix of the gel is influenced by an applied electric field. Typical forms of gel used are agarose and polyacrylamide. The decision to use one over the other typically depends upon the specific need for a certain resolution power (which is greater for poly-acrylamide) or the lack thereof. An agarose gel matrix is usually used in routine electro-phoresis of DNA, such as to resolve PCR reaction products. Protein electrophoresis typically uses polyacrylamide.

The electric field is applied such that the positive electrode occurs on the opposite end of the gel to where the sample is applied (usually the top). For DNA electrophoresis, the nucleic acid samples migrate through the gel matrix towards the positive electrode because DNA has an inherent negative charge, due to the accumulation of phosphate groups in the DNA backbone. The charge of proteins, however, largely depends upon the pH of their environment. To resolve this problem, proteins are usually denatured in the presence of sodium dodecyl sulfate or SDS (a type of soap), which coats the denatured protein molecule and gives it an overall negative charge. This type of protein electrophoresis is referred to as SDS-PAGE.

For SDS-PAGE and normal DNA electrophoresis, after the electric field is applied, the molecules migrate through the gel matrix at rates which depend on their molecular size. Thus, larger proteins and oligonucleotides take longer to weave their way through the maze-like matrix, and thus remain "higher" on the gel, or closer to the sample application point. Smaller proteins and oligonucleotides are better able to pass through the matrix and thus are found "lower" or farther down the gel, closer to the positive electrode. Proteins and DNA molecules separated by electrophoresis are typically detected after separation by staining with fluorescent or colorimetric dyes, such as coomassie brilliant blue (for proteins) and ethidium bromide (for DNA).

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