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Until recently, whole-proteome microarrays for comprehensive studies of protein interactions were mostly produced by individual cloning and cellular expression of very many open reading frames, followed by protein isolation and purification as well as array production. To overcome this cumbersome process, we have developed a method to generate microarrays representing entire proteomes by a combination of multiple spotting and on-chip, cell-free protein expression. Here, we describe the protocol for the production of bacterial protein microarrays. With slight adaptations, however, the procedure can be applied to the proteome of any organism. Expression constructs of each gene are generated by PCR on bacterial genomic DNA followed by a common secondary amplification that is adding relevant regulative elements to either end of the constructs. The unpurified PCR-products are spotted onto the microarray surface. Full-length proteins are directly expressed in situ in a cell-free manner and stay attached to the surface without further action. As an example of a typical application, we describe here the proteome-wide analysis of the immune response to a bacterial infectious agent by characterizing the binding profiles of the antibodies in patient sera.

In situ, Cell-free Protein Expression on Microarrays and Their Use for the Detection of Immune Responses