Quantification of the -translated proteins in cells expressing varied S1 concentration

<p><b>Copyright information:</b></p><p>Taken from "Ribosomal protein S1 influences -translation and "</p><p></p><p>Nucleic Acids Research 2007;35(7):2368-2376.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874662.</p><p>© 2007 The Author(s)</p> ( Strains depleted for S1 contain less his-tagged proteins. Lanes 1 and 2: control strain grown in the absence (−) or presence (+) of IPTG, respectively. Lanes 3 and 4: deleted strain () grown in the presence (+; complementation condition) or absence of IPTG (−; depletion condition), respectively. IPTG induces expression of the extra plasmid-borne copy present in the strains (section ‘Experimental procedures’). Western blot analysis was performed for samples of the cultures used to purify his-tagged protein. Blot was revealed with S1 antibodies mixed with PNPase antibodies to normalize quantification. The S1/PNPase ratio was determined by quantification of the S1 and PNPase signals on a chemi-smart 5000 (Vilbert-Lourmat). Doubling time of each strain is indicated. Amounts of purified his-tagged protein were measured by absorbance (section ‘Experimental procedures’). Percentage of his-tagged proteins was determined after normalization to the amount of total proteins extracted from each strain and strain (lane 1) was used as reference. ( Excess of free S1 affects the accumulation of -translated proteins. Up: schematic representation of the chromosomal fusion used as translational reporter. Western blot analysis was performed as described earlier. β-Galactosidase activities from translational fusion are given in β-galactosidase units corresponding to nanomoles of ONPG hydrolyzed per min and per mg of total protein. The values shown are averages of three independent assays. Presence of the pS1 plasmid strongly decreases β-galactosidase synthesis (20-fold) due to S1 autogenous control. Doubling time is given for both strains. Percentage of his-tagged protein was determined as in , and strain containing the control plasmid pAC, was used as reference