Detergents are organic compounds that have both hydrophilic and hydrophobic characteristics. In their monomeric forms, they are soluble in both water and lipid. Choice of a particular detergent for initial solubilization or for addition after solubilization is somewhat empirical. Educated guesses can be made, however, as to the suitability of a detergent for a particular situation based on its structure and its intended use. Commonly used detergents and their properties are described in appendix 1.
This protocol has been optimized for the solubilization of the T cell antigen receptor utilizing Triton X-100 as the detergent of choice. An optional step is presented in which the ionic detergents sodium deoxycholate (Na-DOC) and sodium dodecyl sulfate (SDS) are added after removal of insoluble material and nuclei. Addition of these reagents facilitates the disruption of interactions between integral membrane proteins and is useful for the purification of individual polypeptides. Their addition, subsequent to removal of the nuclear fraction, avoids problems inherent in nuclear disruption. A similar protocol that has been used for other membrane antigens is provided in unit 8.2.
Phosphate-buffered saline (PBS; appendix2a), ice-cold Triton X-100 lysis buffer with protease inhibitors (see recipe; store at 4°C) 10% sodium deoxycholate (Na-DOC), room temperature (optional; Sigma) 10% SDS, room temperature (optional; Bio-Rad)
Additional reagents and equipment for harvesting and counting cells (appendix 3) NOTE: Carry out all steps in this protocol at 4°C or on ice.
Contributed by Allan M. Weissman
Current Protocols in Immunology (2003) 8.1A.1-8.1A.9 Copyright © 2003 by John Wiley & Sons, Inc.
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