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11.7: Gel Blotting

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    4930
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    Gel blotting is a technique for visualizing a particular subset of macromolecules — proteins, or fragments of DNA or RNA — initially present in a complex mixture. The steps:

    1. Separate the molecules by electrophoresis. This is done in a gel which allows the molecules to migrate under the influence of the electric field.
    2. "Blot" them with a nitrocellulose filter. For unknown reasons, the molecules stick tightly to the filter and will retain their relative positions when flooded with fluid at the next step.
    3. Bathe the filter with a solution containing a "probe": a molecule that will combine specifically with the target molecules; that is, the one(s) you are looking for and carries a mean of visualization, e.g. a radioactive or fluorescent marker.

    Southern Blot

    The diagram illustrates the procedure for detecting DNA fragments containing a particular sequence. DNA is extracted from the cell and is partially digested by a restriction endonuclease. The resulting DNA fragments are separated by electrophoresis and then denatured to form single-stranded molecules (ssDNA). Without altering their positions, the separated bands of ssDNA are transferred to a nitrocellulose filter and exposed to radiolabeled cDNA or RNA. If the probe detects complementary DNA sequences, it will bind to them. The presence of the probe in a particular band is revealed by autoradiography.

    Figure \(\PageIndex{1}\): Southern Blotting

    This procedure was developed by E. M. Southern and the finished product is called a "Southern blot". The same basic procedure can also be used to separate and visualize RNA molecules and protein molecules. As a humorous extension of the term "Southern blot", these have been dubbed "Northern" and "Western" blots, respectively (Table \(\PageIndex{1}\)).

    Table \(\PageIndex{1}\): Summary or Common Gel Blots
    Type of Blot Molecules separated by electrophoresis Probe
    Southern ssDNA cDNA or RNA
    Northern denatured RNA RNA or cDNA
    Western Protein Antibodies

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