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Automated microfluidic protein immunoblotting

Nature Protocols volume 5, pages 1844–1856 (2010)Cite this article

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Abstract

This protocol describes regional photopatterning of polyacrylamide gels in glass microfluidic devices as a platform for seamless integration of multiple assay steps. The technology enables rapid, automated protein immunoblotting, demonstrated in this study for native western blotting. The fabrication procedure is straightforward and requires approximately 3 h from the start of gel photopatterning to completion of native protein western blotting, a substantial time savings over slab-gel immunoblotting. The assay itself requires less than 5 min. Importantly, all assay stages are programmably controlled by a high-voltage power supply and monitored by an epifluorescence microscope equipped with a charge-coupled device camera. Our approach overcomes severe limitations associated with conventional immunoblotting, including multiple steps requiring manual intervention, low throughput and substantial consumption of reagents. We also describe a simple chemical recycling protocol so that glass chips can be reused. The fabrication technique described forms the basis for a diverse suite of bioanalytical tools, including DNA/RNA blotting and multidimensional separations.

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Figure 1: Schematic of the immunoblot protocol.
Figure 2: Immunoblot chip design and operation.
Figure 3: Photopatterning of gels in the immunoblot chip chamber.
Figure 4: Gel precursor solution preparation.
Figure 5: PDMS manifold fabrication.
Figure 6: Running an immunoblot assay.
Figure 7: Glass microfluidic chip regeneration.
Figure 8: Immunoblot assay results.

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Acknowledgements

We acknowledge financial support from the University of California, Berkeley and the QB3/Rogers Family Foundation Award. Facilities and equipment support from UC Berkeley's Biomolecular Nanofabrication Center is also appreciated. PSA samples were a generous gift from D. Peehl, Stanford University. A.E.H. is an Alfred P. Sloan Foundation Research Fellow in chemistry.

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Authors and Affiliations

  1. Department of Bioengineering, University of California, Berkeley, California, USA

    Mei He & Amy E Herr

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  1. Mei He
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Contributions

M.H. designed chips and conducted experiments. M.H. and A.E.H. analyzed data and wrote the paper.

Corresponding author

Correspondence to Amy E Herr.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Microscale immunoblot chip layout and dimensions (unit: mm). Channel depth is 20µm. The closed squares and circles designate areas for etching. The regions within the green and blue boxes are depicted further in Supplementary Figure 2 and Supplementary Figure 3, respectively. (PDF 217 kb)

Supplementary Fig. 2

Green box detail from Supplementary Figure 1 (unit: mm). Channel depth is 20µm. The closed square and triangles designate areas for etching. (PDF 211 kb)

Supplementary Fig. 3

Blue box detail from Supplementary Figure 1 (unit: mm). Channel depth is 20µm. The closed square and triangles designate areas for etching. (PDF 204 kb)

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He, M., Herr, A. Automated microfluidic protein immunoblotting. Nat Protoc 5, 1844–1856 (2010). https://doi.org/10.1038/nprot.2010.142

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