Isolation of domains of the plasma membrane of hepatocytes

Experimental Cell Research
J K PetellD Doyle

Abstract

Several recent studies have demonstrated the ability of techniques based on immunoadsorption to selectively isolate specialized subregions of membranes, termed domains, which are derived from a larger more complex parent membrane like the plasma membrane. The immunoadsorbent is directed against a specific antigen that resides exclusively or predominantly in the membrane domain to be isolated. Thus, a monospecific antibody to the domain-specific antigen is required. In the present study we developed a method employing a modified immunoblotting strategy which could utilize polyspecific antibodies to isolate membrane vesicles derived from a specific membrane domain of the hepatocyte plasma membrane. We also used specific cell surface labeling of the hepatocyte plasma membrane by lactoperoxidase-catalyzed iodination at 4 degrees C and preparation of different sized vesicles by sonication to facilitate isolation of the specific domain. For this study, polyspecific antisera were raised in goats against a membrane fraction, denoted N2u, which is enriched in bile canalicular proteins. This antiserum recognizes, among other antigens, a 110,000 Mr polypeptide previously shown to be localized in the bile canaliculus (J. Cook et al. (1983)...Continue Reading

References

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Citations

Jul 27, 2007·Proteomics·Djuro Josic, James G Clifton

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