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 approach, termed "
Nucleic Acid Protein Programmable Array" (NAPPA), enables the simultaneous expression of
proteins in microarray format without the need for individual
protein purification. 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 preparation of the bacterial cultures in deep-well blocks.