Protein and Nucleic Acid Blotting

Diagram of a blotting experiment. From J. Watson, Molecular Biology of the Gene, 4th ed. (1987), Benjamin/Cum-mings Publishing Company (Redwood City, CA). 

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Explain the purposes of protein and nucleic acid blotting. ... 

Both Parts I and II have been completely rewritten and reflect the many advances in biochemistry-molecular biology theory and techniques. Especially noteworthy have been the technical advances in chromatography (perfusion, FPLC, bioaffinity), electrophoresis (pulsed gel, capillary, nucleic acid sequencing), spectrophotometry (nmr, ms, and diode array detectors), and molecular biology (microsequencing of proteins and nucleic acids, blotting, restriction enzymes). 

Following an electrophoretic run, the band from the tracking dye is often the only visible band. The detection of separated proteins and nucleic acids requires subsequent treatment of the separation pattern for visualization. This treatment may be performed directly on the gel, or may require a blotting step in which the entire separation pattern is transferred onto a thin membrane material. The choice of detection method depends on the concentrations of analytes in the separated zones and whether recovery of the purified sample is required. 

Methods used in EIH may be extended to other techniques where identification and localization of antigens are important the protein and nucleic acid transfer blots (Chapter 16) may be subjected to these methods. As with EIA, epitopes rather than antigens or molecules are recognized by EIH methods. Shared reactivity and crossreactivity (Section 8.6) may, therefore, interfere as in the AA-type assays (Section 2.3). 

H9. Hauber, R., Miska, W., Schleinkofer, L., and Geiger, R., New, sensitive radioactive-free bioluminescence-enhanced detection system in protein blotting and nucleic acid hybridization. J. Biolumin. Chemilumin. 4, 367-372 (1989). 

The ability to discern changes in the regulation of protein expression, not only by the use of antibodies, but also by means of Northern blotting techniques to monitor changes in mRNA levels, and footprinting techniques or gel retardation/ mobility shift assays to determine interaction of regulatory proteins with nucleic acids. 

The ultimate design of the array is up to the investigator. Some experimental designs will place a different specimen in each well, resulting in a multiplex dot-blot format. Alternatively, analytes can be diluted to create a dilution curve for more exact quantifications. A standard curve of biomaterial of known concentration can be included, and if combined with dilution curves of specimen, but utilized for exact quantification of analytes. The platform is compatible with functional assays including kinase assays, enzymatic processes and can be used to study protein-protein, protein-nucleic acid, and nucleic acid-nucleic acid interactions. 

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