Western blot analysis of in vitro reconstituted 30S particles. Reconstituted 30S subunits were formed at 15°C under the following conditions: 16S rRNA:DnaK:DnaJ:GrpE 1:1:1:2. The resulting particles were applied to 10%–40% sucrose gradients (Culver and Noller 1999); the peak was collected and split into two equal fractions. One fraction was directly precipitated, whereas the second was concentrated and washed (with a buffer containing 330 mM KCl) on a Centricon 100 sieving filter prior to precipitation. Equal fractions were probed with monoclonal anti-DnaK antibody from StressGen Biotech. (Lane 1) 50 ng of purified DnaK from StressGen Biotech. (Lane 2) Sucrose-gradient purified reconstituted 30S particle. (Lane 3) Sucrose-gradient purified and Centricon 100 treated reconstituted 30S particle.
