Dolores J. Cahill cahill@molgen.mpg.de. Max-Planck-Institute of Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany. 15 Peltier, J.B. et al. (2000) Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins. Plant Cell 12, 319–342 16 Zachariae,W. et al. (1996) Identification of subunits of the anaphase- promoting complex of Saccharomyces cerevisiae. Science 274, 1199–1204 17 Grossberger, R. et al. (1999) Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex. J. Biol. Chem. 274, 14500–14507 18 Washburn, M.P. and Yates, J.R., III (2000) New methods of proteome analysis: multidimensional chromatography and mass spectrometry. In Proteomics:A Trends Guide (Blackstock,W.P. and Mann, M., eds), pp. 27–30, Elsevier Microarray production is a highly automated process, using either pin-based or microdispensing liquid handling robots to arrange biological samples on a flat surface for multiple analysis. For the generation of DNA arrays, PCR techniques are well established and now play a central role in large-scale genome analysis 1 . The surface used for arraying varies with the application, for example, if large numbers of samples are to be analysed for their interac- tion with the same ligand, DNA is arrayed onto either fil- ter membranes (e.g. nitrocellulose, nylon, polyvinylidene difluoride) or glass slides coated with various reagents (e.g. poly-L-lysine or polyacrylamide). Generation of arrays Similar technology has been used to generate high-density protein arrays 2,3 and micro-arrays 4 (Fig. 1). This method involves using gridding robots that transfer either DNA or the corresponding protein expressed, from microtitre plates onto nylon (Hybond N+, Amersham, for DNA analysis) or polyvinylidene difluoride (Hybond–PVDF,Amersham, for protein analysis) membranes in high-density grids 5 . The spotting robot carries a 384-pin head on a servo- controlled three axis linear drive system which can be positioned with an accuracy of 5 m and produces den- sities of approximately 300 spots/cm 2 . The tip diameter depends on the spotting application but can vary between 150 and 450 m (Ref. 4). In situ expression of recombinant fusion proteins and expression products is then detected using an antibody against a His-tag-containing epitope.Anti- bodies binding non-specifically are then washed away and the filters are incubated with the appropriate labelled sec- ondary antibody and substrate.An image is taken of the fil- ter using a charge coupled (CCD) camera, on exposure of the filter to UV. Custom image analysis software is then used to score positive clones 2 . DNA filters, where clones or PCR products are gridded onto nylon membranes, can be re-used at least 20 times, without significant loss of signal intensity. By contrast, protein arrayed on polyvinylidene 19 Link, A.J. et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682 20 Mintz, P.J. et al. (1999) Purification and biochemical characterization of interchromatin granule clusters. EMBO J. 18, 4308–4320 21 Pandey, A. et al. (2000) Analysis of receptor signaling pathways by mass spectrometry: identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl. Acad. Sci. U. S. A. 97, 179–184 22 Gygi, S.P. and Aebersold, R. (2000) Using mass spectrometry for quantitative analysis. In Proteomics:A Trends Guide (Blackstock,W.P. and Mann, M., eds), pp. 31–36, Elsevier 47 Proteomics: A Trends Guide | Review Protein arrays: a high-throughput solution for proteomics research? High-density DNA and protein arrays are small flat surfaces that allow the simultaneous analysis of thousands of molecular parameters within a single experiment. DNA array technologies have resulted in smaller sample volumes, more efficient analyses and higher throughput. As proteins are more complex and more diverse compared with nucleic acids, development of similar platforms for proteomics has proved difficult. This review outlines current techniques used in the generation and applications of high-density protein arrays, with emphasis on recent developments and applications in proteomics. Proteomics: A Trends Guide July 2000 1471-1931/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1471-1931(00)00006-9