Ohlmeier S, Scharf C, Hecker M
Institute of Microbiology and Molecular Biology, Ernst Moritz Arndt University of Greifswald, Germany.
Electrophoresis. 2000 Nov;21(17):3701-9. doi: 10.1002/1522-2683(200011)21:17<3701::AID-ELPS3701>3.0.CO;2-5.
The genomic sequence of Bacillus subtilis, which is the best studied Gram-positive bacterium, enabled us to obtain a theoretical two-dimensional (2-D) map, demonstrating that about one-third of this proteome has a theoretical alkaline isoelectric point (pI). This represents an important part of the entire proteome, which is not detectable in conventional 2-D gels (pH range 4-7). Sequence analysis revealed that 91% of the ribosomal proteins and a high amount of theoretical membrane proteins should be localized in the alkaline pH range requiring different protein extraction procedures. In order to find the pH range which gives the best resolution results for the alkaline proteins of B. subtilis, immobilized pH gradients (IPGs) with different pH ranges (pH 6-10, 6-11, 4-12, 9-12, and 3-10) were tested and optimized for IPG 4-12. Here we present a version of a first alkaline master 2-D gel for B. subtilis, which is a further complement of the already existing master gel (pH 4-7) in the Sub2D database. Almost 150 spots could be detected and 41 proteins have already been identified.
枯草芽孢杆菌是研究最为透彻的革兰氏阳性菌，其基因组序列使我们能够获得一张理论二维（2-D）图谱，表明该蛋白质组约三分之一具有理论碱性等电点（pI）。这代表了整个蛋白质组的一个重要部分，在传统二维凝胶（pH范围4 - 7）中无法检测到。序列分析表明，91%的核糖体蛋白和大量理论上的膜蛋白应定位在需要不同蛋白质提取程序的碱性pH范围内。为了找到能为枯草芽孢杆菌碱性蛋白提供最佳分辨率结果的pH范围，对不同pH范围（pH 6 - 10、6 - 11、4 - 12、9 - 12和3 - 10）的固定化pH梯度（IPG）进行了测试，并针对IPG 4 - 12进行了优化。在此，我们展示了枯草芽孢杆菌第一张碱性主二维凝胶的一个版本，它是Sub2D数据库中已有的主凝胶（pH 4 - 7）的进一步补充。几乎可以检测到150个斑点，并且已经鉴定出41种蛋白质。
