Protease Digestion of Outer Membranes

1. Outer membrane samples were resuspended to a concentration of 0.5 mg/ml in 0.1 ml sample buffer (125mM Tris-HCl, pH 6.8, 0.5% SDS, 10% (v/v) glycerol, 0.0001% (w/v) bromphenol blue).
2. This was heated at 100ºC for 2 minutes and cooled to 37ºC for protease digestion.
3. Protease was added in 1 µl amounts at the concentrations required and the outer membrane samples were digested for 1 hour at 37ºC.
4. Each sample was then divided in two aliquots.
   • To one aliquot, SDS was added to a final concentration of 2%.
   • The other aliquot was made up to 2% SDS and 10% (v/v) 2-ME, to identify the 2-ME modifiable fragments of protein F.
5. The aliquots were heated at 88ºC and loaded onto a 14% PAGE gel.

The initial solubilization step in 0.5% SDS was found to greatly aid the digestion of the fairly protease-resistant outer membrane proteins.