Fractionation by Ultracentrifugation of Gram Negative Cytoplasmic and Membrane Proteins

Materials and Reagents

1. Bacterial culture media (subject to the needs of the bacterium being studied; for Escherichia coli, Luria Broth or DMEM are suitable)
   
2. Tris base (Thermo Fisher Scientific)
   
3. Triton X-100 (Sigma-Aldrich) (optional)
   
4. Tris-Triton (TT)
   
5. HCl (Hydrochloric acid)
   
6. TT (see Recipes)
   

Equipment

1. Bench top ultracentrifuge (e.g. Beckman Coulter, model: TL-100 ) capable of up to 50,000 rpm for culture volumes of 25 ml or less, and a larger mainframe ultracentrifuge for larger culture volume preparations
   
2. Bench top centrifuge capable of up to 12,225 x g
   
3. Sonicator
   
4. Spectrophotometer
   

Procedure