Blotting
As blotting or blotting is known in the molecular biology to a method for transfer of molecules such as DNA , RNA or proteins to a membrane. The name is derived from the English "to blot" (blot, stain, dab with blotting paper), which alludes to the procedure. The textbook Der Experimenter describes blotting as the "attempt to permanently fix something that has previously been electrophoretically separated in a gel onto a membrane".
Edwin Southern introduced the DNA blotting technique in 1975 called Southern blot . Based on his name, the blotting techniques later developed by George R. Stark were referred to as Northern Blot for RNA and Western Blot for proteins . There are also Southwestern blotting for DNA-protein, Northwestern blotting for RNA-protein and Far Western blotting for protein-protein interaction analyzes.
Common principle
The material to be examined (DNA, RNA or proteins) is separated by means of gel electrophoresis . It is then transferred to a blot membrane (a filter , for example made of nitrocellulose , nylon , PVDF or glass fiber ) (the actual blotting) and fixed there irreversibly. In the next step, an antibody or a radioactively labeled probe (a gene probe for nucleic acids, immunostaining for proteins) is usually added. Gene probes only stick to the sequence sought ( hybridization ). The unbound probes are washed off and the filter is placed on an X-ray film that is exposed by chemiluminescence or autoradiography to display the results of the electrophoresis .
literature
- Thomas Maniatis , Edward F. Fritsch, Joseph Sambrook : Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor NY 1982, ISBN 0-87969-136-0 .
Individual evidence
- ↑ Cornel Mülhardt: The experimenter. Molecular Biology / Genomics. Spektrum, Akademischer Verlag, Heidelberg 2009, ISBN 978-3-8274-2036-7 , p. 75.
- ↑ Edwin M. Southern: Detection of specific sequences among DNA fragments separated by gel electrophoresis. In: Journal of Molecular Biology. Vol. 98, No. 3, November 1975, ISSN 0022-2836 , pp. 503-517, PMID 1195397 , doi : 10.1016 / S0022-2836 (75) 80083-0 .