Characterization of cellular antigens using monoclonal antibodies

This scheme can be regarded as a basis for a characterization study however, it should be noted that some steps may not be universally relevant and that a number of the methods described in this chapter can be adapted and optimized depending on the nature of individual studies. 

The methods used to produce, purify, and characterize monoclonal antibodies are described in detail in Chapters 1-11, and in ref. 1. Initial characterization of a monoclonal antibody is essential before use in experiments and should include  

Cells and tissues must be lysed to release the antigen protein for analysis by immunoblotting and immunoprecipitation. The method of extraction depends upon the subcellular localization of the antigen and the subsequent mode of analysis and detection. The methods described in this section are intended as guides, and can be adapted to optimize the extraction of individual proteins. 

Protocols lA and IB use detergents to achieve whole cell lysis of tissue culture cells and result in solubilization of the plasma membrane, nuclear and organelle membranes, and the cytoskeleton. Tissue samples can also be homogenized in buffers containing detergents using a Polytron homogenizer. 

In Protocol IB, a soluble whole cell extract is produced by lysis in RIPA, a mixed micelle detergent-containing buffer. RIPA buffer results in extraction of proteins without complete denaturation and cellular antigens are maintained in conformations that can be detected by immrmopredpitation (Protocol 14B). How- 

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Characterization of cellular antigens using monoclonal antibodies 265... 

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