Different blotting techniques are used to identify unique proteins and nucleic acid sequences. Southern, northern, and western blot protocols are similar, and begin with electrophoretic separation of protein and nucleic acid fragments on a gel, which are then transferred to a membrane (nitrocellulose membrane, polyvinylidene difluoride (PVDF) membrane, etc.) where they are immobilized. This enables radiolabeled or enzymatically labeled antibody or DNA probes to bind the immobilized target, and the molecules of interest may then be visualized with various methods. Blotting techniques are selected based on the target molecule: DNA, RNA, or protein. (Figure 1, Table 1).
Southern blots are used to determine the identity, size, and abundance of specific DNA sequences. The southern blot protocol begins with DNA extraction from the cells or tissues, which is then enzymatically digested to produce DNA fragments. The fragments are separated by size on an agarose or polyacrylamide gel via electrophoresis. Smaller fragments will migrate farther on the gel than larger ones. Following electrophoresis, the DNA on the gel is transferred to a nylon membrane. The membrane is incubated with a nucleic acid probe that has a sequence homologous to the target sequence and is labeled with radioactivity, fluorescent dye, or an enzyme capable of generating a chemiluminescent signal. Hybridization of complementary sequences occurs during incubation, and the unhybridized probe is removed by washing with buffer. The fully hybridized labeled probe molecules will remain bound to the blot. Detection methods differ based on the probe label; radiolabeled probes are visualized with X-ray film or phosphorimaging, and enzymatically labeled probes are visualized with chemiluminescent substrate. Micro Filter Pad
Northern blots are used to determine the identity, size, and abundance of specific RNA sequences. Northern blot protocols begin with RNA isolation, and separation techniques vary depending on RNA size. Large RNAs are separated by electrophoresis on a formaldehyde agarose gel or glyoxal agarose gel, which prevents normal base paring and maintains RNA in a denatured state. Small RNAs are separated on a denaturing (urea) polyacrylamide gel. The RNA is then transferred from the gel to a nylon membrane which is then incubated with a radioactively or nonisotopically labeled RNA, DNA, or oligodeoxynucleotide probe. The unhybridized probe is removed by washing with buffer. Radiolabeled probes are visualized with X-ray film, and enzymatically labeled probes are visualized with chemiluminescence.
Western blots are used to determine the identity, size, and abundance of specific proteins within a sample. The western blot protocol begins with sample lysate preparation from tissue or cell culture and separation on a polyacrylamide gel via electrophoresis. The separated proteins are then transferred to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The membrane is incubated with a blocking agent to prevent nonspecific binding, followed by incubation with a primary antibody to bind the protein of interest. There are two detection methods, direct and indirect. Direct detection (Figure 2) relies on a labeled primary antibody, whereas indirect detection requires a primary antibody directed against the target protein, and a secondary antibody directed against the immunoglobin class or subclass of the primary antibody’s species (Figure 3). Visualization methods include colorimetric assays in which a colored precipitate is produced, chemiluminescence, and fluorescence.
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