IMPROV METHODS:STRUCT ANALYS OF PROTEIN PRENYL GROUPS & ASSOCD C TERMINAL MODIFI

我们想要研究与各种蛋白质相关的 Cyclin-Cdc28复合体，以Clb2-Cdc28开始。我们已经制作了和 测试了允许高效亲和纯化的标签盒 以及标记蛋白的免疫沉淀。我们已经标记了Clb2，并且 在从不到300毫升的细胞中亲和纯化Clb2后，我们 可以通过银染看到Clb2、Cdc28、另外两个未知的 特异地交配亚粪便蛋白质，而且只有一种 非特异性蛋白质(通过与相同的 从未加标签的对照菌株中纯化)。所有这些蛋白质都是 在标准的SDS-PAGE凝胶上很好地分离，几乎没有 背景染色。我们目前正在一台 更大的规模，我们在CDC28背景和 含有死亡的、过表达的CDC28的菌株以及 野生型CDC28。这个想法是一个Clb2-(激酶死亡的CDC28) 复合体可能会附着在底物上，而且有一些间接的 双杂交实验对此提供了支持。 假设我们得到了特定的带子，我们想要银色染色， 从凝胶中剪下特定的条带，然后提交给 用质谱仪进行鉴定。有三个条件： (1)非标记菌株的对照纯化；(2)实验 标记Clb2、CDC28菌株的纯化；(3)实验 标记Clb2、CDC28、CDC28-激酶死亡菌株的纯化。 可能的底物可能是第三种特定的。 条件，而其他种类的相关蛋白质可能被发现在 第二和第三个条件。

We would like to look at proteins associated with various cyclin-Cdc28 complexes, starting with Clb2-Cdc28. We have made and tested a tagging cassette that allows efficient affinity purification and immuno-precipitation of tagged proteins. We have tagged Clb2, and after affinity purification of Clb2 from less than 300 ml of cells, we can see by silver staining Clb2, Cdc28, two other unknown but specifically copurifying sub-stochiometric proteins, and just one non-specific protein (as judged by comparison with the same purification from an untagged control strain). All these proteins are well separated on standard SDS-PAGE gels, and there is little background staining. We are presently doing the experiment on a larger scale, and we are doing it in both a CDC28 background, and in a strain that contains a kinase-dead, over-expressed Cdc28 as well as wild-type Cdc28. The idea of this is that a Clb2-(kinase dead Cdc28) complex might hold on to substrates, and there is some indirect support for this from two-hybrid experiments. Assuming we get specific bands, we would like to silver stain, cut the specific bands out of the gel, and submit them for identification by mass spectrometry. There would be three conditions: (1) control purification from an untagged strain; (2) experimental purification from a tagged Clb2, CDC28 strain; (3) experimental purification from a tagged Clb2, CDC28, cdc28-kinase dead strain. Potential substrates would presumably be specific to the third condition, while other kinds of associated proteins might be found in both the second and third conditions.