Blotting Information and Courses from MediaLab, Inc.
These are the MediaLab courses that cover Blotting and links to relevant pages within the course.
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| Resurgence of Electrophoresis Traditionally most clinical laboratory electrophoresis utilizes methods that separate and identify proteins in serum, urine, CSF, and some other body fluids. Most studies are for detecting serum protein abnormalities and gathering more information about gammopathies.In recent years, there has been a resurgence in electrophoresis use and methods. Development of automated methods has enhanced this. The evolution of numerous molecular diagnostic investigations and research in proteomics have also augmented electrophoresis.Applications of two-dimensional electrophoresis discussed the use of electrophoresis in proteomics. Electrophoresis and molecular diagnostics, blotting techniques, and current uses of CE in molecular diagnostics will be discussed now. | View Page |
| Electrophoresis and Molecular Diagnostics Because of ionized phosphate groups, both DNA and RNA will migrate in an electrical field with an appropriate buffer. They are negatively charged and will migrate to the anode. The speed of migration and separation achieved is based upon size with smaller molecules traveling faster. The shape of macromolecules, type of support medium, and electrophoresis method also vary the separation results. The isolated nucleic acid can be single-stranded or double-stranded and can fold into other structures. AGE, PAGE, and CE are the most common electrophoresis methods used in analysis of nucleic acids. Pulsed electric fields are needed to separate large fragments. The electrophoresis employed in blotting techniques enhance these discrimination techniques. | View Page |
| Blotting Techniques Blotting techniques were developed to discriminate fragments of nucleic acids. These techniques involve several processes; electrophoresis is one of the processes and is used to separate fragments of DNA and RNA. In Southern blotting (named after Edward Southern) restriction enzymes cut fragments of DNA are separated by AGE or PAGE, transferred to a membrane or blot, and visualized by hybridization with labeled probes.Northern blotting (not named after an inventor but by analogy to Southern blotting) separates RNA. RNA molecules are shorter and have defined lengths; cutting by restriction enzymes is not required. Denaturing conditions are required because of RNA secondary structures. After membrane blotting, the separated types of RNA are visualized with staining or labeled probes.Western blotting (again not named after an inventor but by analogy to Southern blotting)does not separate nucleic acids; it separates proteins in a mixture. The proteins are usually separated with PAGE, transferred to the membrane and visualized with a labeled antibody against the proteins of interest. | View Page |