Abstract
Publisher Summary This chapter discusses the prepration of monoclonal antibodies and site-detected polyclonal antibodies against the lac Permease of E.coli. The lac permease (that is, lac carrier protein) of Escherichia coli is an intrinsic membrane protein, the product of the lac Y gene that catalyzes the translocation of β -galactosides with hydrogen ion in a symport or cotransport reaction. The permease can be solubilized from the membrane, purified to homogeneity, reconstituted into phospholipid vesicles, and shown to catalyze all of the transport reactions typical of the native membrane with comparable activities and kinetic properties. To test the predictions, to determine the orientation of the permease in native and reconstituted membranes, and to obtain information regarding structure or function relationships, monocionai antibodies (Mabs) against purified lac permease and polyclonal antibodies directed against synthetic peptides corresponding to portions of the permease have been utilized. The preparation of Mabs against purified immunogens such as lac permease is relatively straightforward. Advantageous aspect of Mabs is their potential for inhibiting catalytic activity. However, the most significant advantages of site-directed polyclonal antibodies are that they are directed against a predefined epitope and that their generation takes less time and less effort than Mabs.
Published Version
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