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A dot blot (or slot blot) is a technique in molecular biology used to detect biomolecules, and for detecting, analyzing, and identifying proteins. It represents a simplification of the northern blot, Southern blot, or western blot methods. In a dot blot the biomolecules to be detected are not first separated by electrophoresis. Instead, a mixture containing the molecule to be detected is applied directly on a membrane as a dot, and then is spotted through circular templates directly onto the membrane or paper substrate. This differs from the western blot because protein samples are not separated electrophoretically. This is then followed by detection by either nucleotide probes (for a northern blot and southern blot) or antibodies (for a western blot). The technique offers significant savings in time, as chromatography or gel electrophoresis, and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target biomolecule. Furthermore, if two molecules of different sizes are detected, they will still appear as a single dot. Dot blots therefore can only confirm the presence or absence of a biomolecule or biomolecules which can be detected by the DNA probes or the antibody. Because dot blot does not require complicated instrument to operate, lots of dot blot assays are developed using antibodies with high specificity to detect different protein targets. Dot blot is also used to evaluate or screen the effectiveness of the antibodies. == Protocol == Below is just a general guide line for a antigen-antibody-antibody dot blot assay protocol. Specific concentration need to be determined for each assay. Other type of dot blot assay, such as antibody-antigen-antibody can be performed in a similar fashion. 1. spot 1-2 microliter of antigen on to a piece of membrane, let air dry for 30 min or longer; 2. incubate with blocking buffer for 30 min -2 hr; 3. rinse with rinsing buffer, 3x5 min; 4. incubate with primary antibody, 30 min - 2 hr; 5. rinse with rinsing buffer, 3x5 min; 6. incubate with enzyme-labeled secondary antibody, 30 min - 2 hr; 7. rinse with rinsing buffer, 3x5 min; 8. add enzyme substrate, wait 5-10 min; 9. detect by eye or with colorimetric or chemiluminescent imaging system. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「dot blot」の詳細全文を読む スポンサード リンク
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