FIG 6.

Membrane flotation of CtsP-FLAG. Soluble and membrane fractions of DRH212, cts mutants (ΔctsP, ΔctsE, and ΔctsX), and E. coli containing the fusion protein CtsP-FLAG expressed on pECO102 were obtained. The membrane fractions were resuspended in a sucrose gradient to separate the membranes from the insoluble material and subjected to ultracentrifugation, and fractions were taken moving sequentially down the gradient (numbered 1 to 6, where 1 is the top of the gradient and 6 contains the insoluble proteins). These fractions were separated via 12% SDS-PAGE. The presence of the CtsP-FLAG fusion protein was detected by immunoblotting using a monoclonal antibody against the FLAG epitope. S, soluble fraction; M, membrane fraction.