Immunochemical purification of probe‐labeled plasma membrane proteins: An approach to the molecular anatomy of the cell surface

Abstract

The probe 2,4,6‐trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labelling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate‐polyacrylamide gels of probe‐labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus‐transformed cells. Moreover, the transformed cells display a nonvirion component of 80–100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37°C but not at 41°C, the expression of this component is related to the expression of the transformed phenotype.