Fig 4. Co-localization of TcpB with purified TCP^H181A.

(A) Coomassie-stained SDS gel of TCP^H181A purified from V. cholerae RT4225, which has a mutation encoding a His181Ala substitution in the major pilin, TcpA. The protein concentration of the solution was found by spectrophotometry (A₂₈₀) to be 10 mg/ml. The lane was overloaded with 90 μg of protein to show the purity, estimated at >95%. The positions of mass markers are indicated. The protein band at ~50 kDa is not likely to be TcpB (46 kDa), which based on the TcpA:TcpB stoichiometry determination (see below) is present in quantities too small to detect by Coomassie stain. (B) Immunoblots of pilins and pilus assembly components in purified TCP^H181A and whole cell culture. For the purified TCP^H181A samples, the total amount of protein loaded is indicated; for the whole cell culture samples, 25 μl was loaded into each well. Blots were probed with antibodies against TcpA and TcpB; TcpC and TcpE were also probed to assess contamination from outer and inner membranes, respectively. All blots were developed for 2 minutes.