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 a method for expression of the desired proteins in situ on the printed slides.