Immunoblotting technique

Baroni, M. V., Chiabrando, G. A., Costa, C., and Wunderlin, D. A. (2002). Assessment of the oral origin of honey by SDS-PAGE immunoblot techniques. /. Agric. Food Chem. 50, 1362-1367. 

Actually doing tissue printing is the best way to understand its simplicity (Fig. 1). Wide variations in the technique are possible and can be developed easily and quickly according to need. The following protocol for using tissue prints to detect tissue distribution of IAA-protein con-juguates in okra fruit details the immunoblotting technique as follows. 

Separation of a mixture of proteins by electrophoretic techniques such as polyacrylamide gel, SDS polyacrylamide or iso-electric focusing usually results in a complex pattern of protein bands or zones. Interpretation of the results often involves a comparison of the patterns of test and reference mixtures and identification of an individual protein, even using immunoelectrophoresis (Figure 11.15), is very difficult. However, specific proteins can often be identified using an immunoblotting technique known as Western blotting. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein. 

Evans, J. P, Wickremasmghe, R. G, and Hoffbrand, A. V. (1987) Detection of tyrosine protein kinase substrates in fresh leukemia cells and normal blood cells using an immunoblotting technique. Leukemia 1, 782—785... 

The dimension of an antibody library, typically defined as the number of clones bearing a suitable selectable marker (antibiotic resistance) and, containing the full-size antibody gene (detectable by PCR screening 32), does not necessarily correspond to the functional dimension of a library, which requires that the clones express properly folded antibodies. An approximation for the determination of the functional dimension of a library consists of determining what percentage of clones expresses antibodies, for example, using immunoblot techniques. 

Key Words Western blotting immunoblotting electrophoresis 2D electrophoresis immunoaffinity identification immunoblotting techniques. 

Immunoblotting techniques involve the identification of a protein target via antigen-antibody-specific reactions. Proteins are typically separated by electrophoresis in polyacrylamide gels, and then transferred ( blotted ) onto chemically resilient membranes (e.g., nitrocellulose, polyvinylidene difluoride) where they bind in the pattern they took in the gel. The membrane is overlaid with a primary antibody directed to the specific target, then with a secondary antibody (anti-immunoglobulin) labeled with radioisotopes, enzymes or other marker compounds. 

Poxton, I. R. (1990) Immunoblotting techniques. Curr. Opin. Immunol. 2, 905-909. 

Wasowicz, M., Viswanathan, S., Dvomyk, A., Grzelak, K., Kludkiewicz, B., and Radecka, H. 2008. Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium. Biosens Bioelec 24 284—289. 

Microproteins 2-Dimensional electrophoresis immunoblotting techniques Nephelometry, turbidimetry... 

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