Question on the Northern, Southern, and Western Blotting technique?

Hi Darkbands,

The first step in a Western blotting procedure is to separate the macromolecules using gel electrophoresis. After electrophoresis, the separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Next, the membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the membrane. Most commonly, the transferred protein is complexed with an enzyme-labeled antibody as a probe. An appropriate substrate is then added to the enzyme and together they produce a detectable product such as a chromogenic precipitate on the membrane for colorimetric detection. The most sensitive detection methods use a chemiluminescent substrate that, when combined with the enzyme, produces light as a byproduct. The light output can be captured using film, a CCD camera or a phosphorimager that is designed for chemiluminescent detection. Alternatively, fluorescently tagged antibodies can be used, which are directly detected with the aid of a fluorescence imaging system. Whatever system is used, the intensity of the signal should correlate with the abundance of the antigen on the membrane.

Hope this helps answer part of your question.