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When Microarrays Go West(ern)

Western blotting was first introduced in 1979 and has become a routine technique for protein analysis in laboratories worldwide. While western blotting can be highly specific, it is very low throughput as a technique and requires large amounts of sample and expensive antibodies. Furthermore, blots are extremely labour intensive and require a high level of skill on the part of the researcher. The use of fluorophore-conjugated antibodies and advances in digital imaging have overcome this somewhat, allowing for multiplex analysis and quantitation. Even with these advances, however, analysis is still limited to two or three proteins at a time.

The Richard Jones group at the University of Chicago recently published a paper in Nature Methods describing microwestern arrays (MWA), a technique which maintains the specificity of western blotting but scales up the throughput to the level of DNA microarrays.

Firstly, cell lysates are printed onto gels using the layout and sizing of a 96 well microtiter plate.  The gels don’t require a stacking layer due to the use of a special buffer during the electrophoresis process. After this, the gel is divided up so that each set of lysates can be probed with a different primary antibody. This is followed by incubation with a dye-labelled secondary antibody and finally analysis using an infrared (IR) fluorescence scanner.

This technique allows a high level of throughput, enabling 6 samples to be exposed to 192 antibodies. Furthermore, it drastically reduces the cost of protein analysis, as each measurement requires very small amounts of protein (~250 ng) and detection antibodies (16 ng). The analysis using an IR fluorescence scanner allows for accurate quantification and rapid interpretation of results.

This is an exciting technique, as it allows the examination of a large group of proteins at one time. The authors of the Nature Methods paper demonstrated the potential of the technology by examining the behaviour of proteins in a cell line with elevated amounts of epidermal growth factor receptor. They found that EGFR simultaneously activated a number of other receptors in the cell – a possible explanation for tumour resistance to cancer treatments [1, 2].

It was suggested a few years ago that the western blot should be “retired”[3]. The development of the microwestern array demonstrates how older techniques can be combined with new technology, thus greatly expanding the potential scope of research.

References:

  1. Ciaccio, M.F., et al., Systems analysis of EGF receptor signaling dynamics with microwestern arrays. Nat Meth, 2010. 7(2): p. 148-155.
  2. Communications, T.U.o.C.M. Illuminating protein networks in one step. 2010.
  3. Mann, M., Can Proteomics Retire the Western Blot? Journal of Proteome Research, 2008. 7(8): p. 3065-3065.

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