Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.
Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.
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DOI：
10.1093/nar/gni079
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发表时间：
2005-05-12
影响因子：
14.9
通讯作者：
Mermod N
中科院分区：
文献类型：
--
作者：
Egener T;Roulet E;Zehnder M;Bucher P;Mermod N
DNA结合蛋白的功能不仅受其丰度的控制，而且主要受其与DNA和蛋白质靶点相互作用的活性水平的控制。此外，这类转录因子对其靶序列的亲和力通常由辅因子和/或修饰控制，这些辅因子和/或修饰不容易从生物样品中评估。在这里，我们描述了一个可扩展的方法，用于监测蛋白质-DNA相互作用的微阵列表面。这种方法被设计用于确定粗细胞提取物中蛋白质的DNA结合活性，补充常规表达谱阵列。DNA的酶标记能够使蛋白质与微阵列的结合直接标准化，从而允许估计相对结合亲和力。使用覆盖一系列亲和力的DNA序列，我们表明，新的基于微阵列的方法产生的结合强度估计类似于低通量凝胶迁移率变化测定。微阵列也具有高灵敏度，因为它允许检测来自乳腺癌细胞的罕见DNA结合蛋白，即人类肿瘤抑制因子AP-2。因此，这种方法介导了DNA结合蛋白活性的精确和稳健的评估，并将目前的DNA结合测定带到高通量水平。
The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein–DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.
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影响因子：
46.9
作者：
Roulet, E;Busso, S;Bucher, P
通讯作者：
Bucher, P
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