![]() Northern blot is a further development of Southern blot for detecting specific RNA sequences. Other labeling systems including fluorescent and chemiluminescent reagents have been applied as well. For instance, target DNA/radioactive-labeled probe hybrid can be detected by radioautography or fluorography. After blotting (transfer and immobilization), the membrane carries a reproduction of the gel pattern, allowing target DNA sequences on the membrane to be detected selectively by hybridization with labeled RNA or DNA probes (complementary to the target sequence) and subsequent visualization according to the nature of labels. ![]() Notably, the post-transfer immobilization step is not required when the combination of positively charged nylon membrane and alkaline transfer buffer is applied, since the binding between DNA and positively charged membrane is irreversible under alkaline conditions. The fragments are then transferred from the gel to a membrane (either nitrocellulose or nylon) by capillary action, followed by immobilization via baking (for nitrocellulose) or ultraviolet irradiation (for nylon). In most applications, the mixture of restriction DNA fragments is size-separated by agarose gel electrophoresis. Southern blot is used to detect specific DNA sequences in a complex DNA mixture (digested genomic DNA resulting from restriction endonuclease cleavage). In particular, I would like to emphasize the smartness of the blotting step in a blotting technique ( Figure 1), which truly enables the subsequent detection of target molecules in a convenient way. The present article is aimed to provide a brief introduction to the blotting technique process for readers since the direction-named techniques can be confusing, especially for readers from outside the biotechnology field. Blotting compass containing the main three techniques (Southern blot, northern blot, and western blot) and some other relative techniques using the direction-oriented naming system. It is worth mentioning that, the directional word that precedes “blot” should be lowercase except for Southern blot, as only Southern blot is named after a person (Edwin Southern). Other modified blotting techniques such as middle-eastern blot, eastern blot, far-eastern blot, northwestern blot, southwestern blot, and far-western blot, following the direction-oriented naming tradition, have been subsequently developed based on the classical three techniques ( Figure 2). The main three blotting techniques are Southern blot (developed by Edwin Southern, 1975), northern blot (developed by James Alwine, David Kemp, and George Stark, 1977), and western blot (developed by Harry Towin, Theophil Staehelin, and Julian Gordon, 1979), which are used for the detection of DNA, RNA, and protein, respectively. In general, a blotting technique is accomplished by: 1) size-based separation of molecule mixture from samples by gel electrophoresis 2) transfer and subsequent immobilization of size-fractionated molecules from the gel onto a membrane support, allowing the formation of a faithful and stable replica of the gel pattern on the membrane 3) specific hybridization of labeled probes to the molecules of interest (target molecules) immobilized on the membrane 4) visualization of the labeled probe/target molecule complex through imaging, allowing identification of the presence, size, and amount of the target molecule by the presence, location, and thickness of a specific band on the image, respectively ( Figure 1). ![]() In conclusion, not only is blotting a smart strategy for enabling the detection of molecules of interest but also the concept of blotting plays an important role in biotechnology.īlotting is a classical investigative technique used to detect macro-biomolecules (such as nucleic acids and proteins) of interest from samples (mixture of molecules). Specifically, the blotting step allows for fixation of size-separated molecules on the membrane, and permits subsequent hybridization of the target molecule to labeled probe, collectively allowing target molecules to be detected by imaging. Particularly, the smartness of the blotting step in a blotting technique is emphasized. The present article briefly introduces blotting techniques for readers, since the direction-named techniques are confusing for people beyond the biotechnology field. The technique is generally composed of four steps: 1) gel electrophoresis-based separation of molecules, 2) blotting (transfer and immobilization) of separated molecules from the gel onto the membrane, 3) specific hybridization of probes to target molecules on the membrane, and 4) visualization of the probe/target molecule complex. ![]() Blotting is a classical technique for detecting macro-biomolecules of interest from a mixture of molecules.
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