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

我们想看看与各种 细胞周期蛋白-Cdc 28复合物，从Clb 2-Cdc 28开始。 我们已经做了， 测试了允许有效亲和纯化的标记盒 和标记蛋白的免疫沉淀。 我们标记了Clb 2， 从少于300 ml的细胞中亲和纯化Clb 2后，我们 通过银染可以看到Clb 2，Cdc 28，另外两个未知但 特别是共纯化亚化学计量的蛋白质， 非特异性蛋白质（通过与相同的 从未标记的对照菌株纯化）。 所有这些蛋白质 在标准SDS-PAGE凝胶上分离良好， 背景染色。 我们目前正在做一个实验， 更大的规模，我们在CDC 28的背景下， 含有激酶死亡的、过表达的Cdc 28以及 野生型Cdc 28。 这一想法是，Clb 2-（激酶死亡Cdc 28） 复合物可能会抓住底物，并且存在一些间接的 双杂交实验的支持。 假设我们得到了特定的条带，我们想用银染色， 从凝胶中切出特定的条带，并将其提交给 质谱鉴定。 有三个条件： (1)从未标记的菌株进行对照纯化;（2）实验性的 从标记的Clb 2，CDC 28菌株中纯化;（3）实验 从标记的Clb 2、CDC 28、cdc 28-激酶死亡菌株纯化。 潜在的底物可能是第三种特有的 条件，而其他类型的相关蛋白质可能会发现， 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.