Figure 3

H-NS-transposase cross-linking. (A) and (B) Transpososomes assembled in the presence of H-NS were subjected to cross-linker (EDC/NHS) treatment as described in Methods. The resulting H-NS-transpososome was purified from a native polyacrylamide gel and proteins eluted from this gel slice were subjected to Western blot analysis using a monoclonal antibody to the T7 gene 10 epitope present on the N-terminus of transposase. Subsequently, the blot was stripped and re-probed with a polyclonal antibody to H-NS. Signal from the marker lane was used to align each pair of blots. In (A) EDC/NHS cross-linking was also carried out on reactions containing purified transposase and H-NS in the absence of DNA. In (B) aliquots of cross-linked H-NS-transpososome were treated with micrococcal nuclease prior to Western blot analysis. T'some, gel-purified H-NS-transpososome; T'ase, purified transposase protein; 'a' and 'b', products inferred to contain transposase cross-linked to H-NS.