Western blotting technique sections

Blotting techniques. The Southern and Northern blots are used to separate and characterize DNA and RNA, respectively. The Western blot, which uses antibodies to characterize proteins, was described in Section 4.3.4. 

Similarly, RNA molecules can be separated by gel electrophoresis, and specific sequences can be identified by hybridization subsequent to their transfer to nitrocellulose. This analogous technique for the analysis of RNA has been whimsically termed Northern blotting. A further play on words accounts for the term Western blotting, which refers to a technique for detecting a particular protein by staining with specific antibody (Section 4.3.4). Southern, Northern, and Western blots are also known respectively as DNA, RNA, and protein blots. 

Many ancillary techniques have been developed in recent years, but several stand out in their ability to extend the usefulness of 2D6EL. This final section will discuss four of these Western blotting, antibody production from gel spots, and metabolic labelling to determine amino acid composition and tyrosine-specific phosphorylation. 

In contrast, when using fluorescence immunocytochemistry to label proteins in tissue sections, the absolute relationship of fluorescence intensity to protein abundance can only be determined through conducting rigorous control experiments, and thus linearity should not be presumed. With that said, because CCD and photomultipHer tube detection systems are linear across a broad range of fluorescence intensities [36], quantitative fluorescence microscopy has been used in numerous smdies to measure relative changes in protein level [33, 35, 36]. In a system where the expression of a protein is only effected in a subpopulation of cells (e.g., GAD67 expression is reduced in a subset of PV+ interneurons in schizophrenia—see ref. 45), quantitative fluorescence immunohistochemistry will be more informative than techniques (e.g.. Western blot) that treat the tissue as a whole. 

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