Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins

Protease Protection Assays. After translation, reactions were chilled on ice and 10-~1 aliquots were diluted to 50 t~l in buffer 1. Microsomes were stabilized by the addition of CaC12 to a final concentration of 10 #M and incubated for 10 rain. Either water or proteinase K (predigested for 30 rain at 30°C) was added to final concentrations of 0.1 and 0.5 mg/ml, or trypsin (predigested in the same way) was added to final concentrations of 0.5 and I mg/ml from 10× stock solutions in water, respectively. In addition, where indicated, NP-40 was added to a final concentration of 1% from a 10% (wt/vol) stock in water. All digests were incubated for 1 h on ice. Protease activity was blocked by the addition of 5 mM DFP, 1% SDS (final concentrations), and boiling for 5 rain, following the method described by Chavez and Hall (1992) . Samples were then diluted 10 times with a buffer (buffer 2) containing 150 mM NaCI, 1% NP-40, 0.5% deoxycholate, and 50 mM Tris, pH 7.5, chilled on ice, and processed for immunoprecipitation analysis, as described below. Immunoprecipitated polypeptide fragments were analyzed on special 20% SDS gels, allowing the resolution of small polypeptide fragments (Thomas and Kornberg, 1975) .