Link Andrew J, Labaer Joshua
Vanderbilt University School of Medicine, Nashville, TN 37232-8575, USA.
CSH Protoc. 2008 Nov 1;2008:pdb.prot5061. doi: 10.1101/pdb.prot5061.
INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes antibody detection of the arrayed proteins.
引言
功能蛋白质组学能够使用高通量（HT）方法在体外研究蛋白质活性。蛋白质微阵列是首选方法，因为它们可以同时展示多种蛋白质，并且评估功能仅需要少量反应体积。蛋白质微阵列通常用于：（1）测量样品中许多不同分析物的丰度，或（2）研究点样在阵列上的许多蛋白质的功能或特性。靶向蛋白质微阵列通常通过将蛋白质表达、纯化并以非常紧密的空间密度点样到固体表面来生成。另一种方法是在阵列表面原位翻译蛋白质。该方法使用无细胞提取物将DNA转录并翻译成蛋白质，然后原位捕获，从而将基因的cDNA拷贝转化为所需的靶蛋白。不是在阵列的每个特征处打印蛋白质，而是将产生所需蛋白质的相应基因的cDNA分子固定到阵列上。玻片的化学处理和DNA分离可以提前进行并储存。然后可以打印质粒DNA以制备NAPPA玻片，其可以干燥储存以供使用。对于实验，将NAPPA玻片进行表达，然后使用抗体和染色剂检测蛋白质和DNA。本方案描述了对阵列蛋白质的抗体检测。
