Cell子刊！！Wes研究链霉球菌培养基中菌丝发育相关蛋白

       近日，英国诺里奇研究院分子微生物学系和美国杜克大学医学院生物化学系的科学家利用Wes在MolecularCell（IF：14.548）发表文章：c-di-GMP Arms an Anti-s to Control Progression of Multicellular Differentiation in Streptomyces，Molecular Cell 77, 1–14.

研究摘要

      链霉菌是主要抗生素来源，在复杂的发育生命周期中，与孢子的形成同时产生。信号分子c-di-GMP结合BldD，以控制发育的开始。在本文中，c-di-GMP通过武装一种新型的抗-S（RsiG）结合并隔离形成孢子的特异性S因子（sWhiG）来控制生殖菌丝分化为孢子。本文介绍了RsiG-（c-di-GMP）2-sWhiG复合物的结构：一个不寻常的，部分插入的c-di-GMP二聚体与RsiG-sWhiG界面处的结合。RsiG在不存在sWhiG的情况下，采用在螺旋线圈的每个螺旋上重复的新颖E（X）3S（X）2R（X）3Q（X）3D序列，与c-di-GMP结合。进一步的研究表明c-di-GMP对于RsiGYZsWhiG是必不可少的。这些发现揭示了s-抗-s复合物形成的控制机制，并揭示了c-di-GMP作为链霉菌发育的主要整合者。Wes全自动定量western blot
       本文利用Wes全自动定量Western blot技术，对孢子形成发育过程中的sWhiG蛋白进行定量。样本来源：链霉菌培养DNB培养基，离心取菌丝球粒，再进行超声裂解。裂解液终浓度0.7 mg/mL，取3μl进行Wes定量western blot检测。（具体方法见下文）

       本文后续利用Wes，定量检测与WTrsiG等位基因和rsiG*等位基因互补的rsiG突变体中的RsiG蛋白水平。

       本研究样本处理及实验条件如下：

       For analysis of protein levels, mycelial pellets originating from 5 mL of culture grown in DNB were resuspended in 2 mL of ice-cold wash buffer (20 mM Tris pH 8.0, 5 mM EDTA). Samples were centrifuged at 13,000 rpm for 1 min at 4C. The supernatant was removed, and the pellet resuspended in 0.4mL of sonication buffer (20mMTris pH 8.0, 5mMEDTA, 1 x EDTA-free protease inhibitors [Roche]). Samples were sonicated at 4.5-micron amplitude for 7 rounds of 15 s, separated by 15 s of rest on ice. Lysed samples were then centrifuged at 13,000 rpm for 15 min at 4C in order to remove cell debris. Total protein concentration was determined using a Bradford assay (Biorad). For analysis of RsiG or RsiG* levels, cell lysates were diluted to a final concentration of 0.7 mg/mL and loaded in triplicate into a microplate (ProteinSimple #043-165), along with a polyclonal anti-RsiG antibody raised in rabbit (Cambridge Biosciences) diluted 1:50. For analysis of sWhiG levels, cell lysates were diluted to a final concentration of 0.2 mg/mL and loaded in triplicate, along with a polyclonal anti- sWhiG antibody raised in rabbit diluted 1:50. For both cases, protein levels were analyzed using the automated Western Blotting machine Wes (ProteinSimple, San Jose, CA) with the Wes-Rabbit (12 to 230 kDa) Master kit according to the manufacturer’s instructions.

       更多Wes/Jess系列定量western blot技术，请联系ProteinSimpleZG

（来源：ProteinSimple）