Fig. 4.

Detection of α- and β- subunits on RK2 plasmid DNA in crude cell extract. Nucleoprotein complexes were formed with the use of an E. coli C600 crude cell extract, and gel filtration was carried out as described in SI Materials and Methods. Reaction mixtures, prepared as for in vitro replication (FII), contained pKD19L1 or non-oriV pUC18 (2.3 nM), wt TrfA (120 nM), TrfA F138A (120 nM), TrfA ∆LF (120 nM), or no TrfA (as indicated). After a 30-s incubation, reactions were cross-linked by the addition of formaldehyde (0.01%) and incubated for an additional 10 min. Fractions collected (40 μL) during gel filtration were applied to an ELISA MaxiSorp plate (Nunc). (A–F) The presence of α or (G–I) β was detected using an immune-enzymatic assay with anti–α- or anti–β-antibodies, respectively, and A at 450 nm was measured. Nucleoprotein complexes were eluted in void fractions (marked with gray). (Top) Presence of DNA was analyzed using agarose electrophoresis.