Purification of Messenger RNA by Polysome Isolation with Monoclonal Antibodies
In this chapter we discuss the use of monoclonal antibodies to enrich the mRNAs coding for low-abundance proteins by polysome immunopurification. In this technique polysomes (which consist of mRNA, ribosomes, and nascent polypeptide chains) are purified by immunoaffinity chromatography with antibodies that recognize antigenic determinants present on the nascent chains. While there have been numerous reports of the use of polyclonal antisera for this purpose, we are aware of only a few examples of the use of monoclonal antibodies, apart from our own work.1,2 Since the preceding chapter by Kraus addresses the use of polyclonal antisera, we shall focus on the differences between monoclonal antibodies and polyclonal antisera with respect to this application. Our own experience in cloning the mRNA for p97, a 97,000-dalton cell surface glycoprotein of human melanoma, which we identified by using monoclonal antibodies,3,5 will be described in some detail. The protein p97 is present in most human melanomas, but it is found in only trace amounts in normal adult tissues.6-8 It is representative of many other low-abundance cell-surface proteins in that it accounts for only 0.02% of total cell protein even in the cell line expressing the largest amounts of the antigen, and it is reasonable to assume that its mRNA comprises a similar proportion of the cellular mRNA.
KeywordsPolyclonal Antiserum Translation Product Immunoaffinity Chromatography Normal Adult Tissue Methyl Sulfonyl Fluoride
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