Analysis of complementation of LJ14 () harboring pTrc-RRF (sectors 1), pBR- (sectors 2) and pTrc99C (sectors 3)

<b>Copyright information:</b>Taken from "Evidence for a role of initiation factor 3 in recycling of ribosomal complexes stalled on mRNAs in "Nucleic Acids Research 2005;33(17):5591-5601.Published online 30 Sep 2005PMCID:PMC1240113.� The Author 2005. Published by Oxford University Press. All rights reserved Overnight cultures were streaked on LB agar ampicillin plates, and incubated for 36 h at the indicated temperatures. Immunoblot analysis of IF3 levels in total cell extracts (?15 �g) of LJ14 harboring pTrc-RRF (lane 1), pBR- (lane 2) or vector alone, pTrc99C (lane 3). In lane 4, ?0.2 �g IF3 was analyzed as marker. A Coomassie blue stained gel is shown in the above panel (i) as a control for equal loading of total proteins. Band corresponding to IF3 is indicated in the immunoblot in the lower panel (ii). Analysis of growth of transformants of various derivatives of LJ14 () and STL1670 in LB broth containing ampicillin, at 30�C (left) and 37�C (right). Saturated cultures grown at 30�C were inoculated (0.1%) into LB broth containing ampicillin and the growth was monitored at 595 nm at regular time intervals. Details of various strains are as shown in the panels.

<b>版权信息：</b>本文内容取自《核酸研究（Nucleic Acids Research）》2005年；33(17)：5591-5601，于2005年9月30日在线发表。PMCID:PMC1240113。© 作者2005年。由牛津大学出版社出版，保留所有权利。将过夜培养物划线接种于含氨苄青霉素的LB琼脂平板上，于指定温度下培养36小时。对携带pTrc-RRF（泳道1）、pBR-（泳道2）或空载体pTrc99C（泳道3）的LJ14菌株总细胞提取物（约15 μg）中的起始因子3（Initiation Factor 3, IF3）水平进行免疫印迹分析。泳道4中加入约0.2 μg IF3作为标记对照。上图（i）为考马斯亮蓝染色凝胶，用于验证总蛋白上样量的均一性。下图（ii）的免疫印迹中标注了对应IF3的条带。对LJ14的各类衍生菌株以及STL1670菌株在含氨苄青霉素的LB肉汤中的生长情况进行分析，分别在30℃（左图）和37℃（右图）下开展实验。将在30℃下培养至饱和的培养液以0.1%的接种量转接至含氨苄青霉素的LB肉汤中，定期在595 nm波长下监测吸光度以追踪生长状态。各菌株的详细信息如各图中标注所示。