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  • Title: Miniaturized capillary isoelectric focusing in plastic microfluidic devices.
    Author: Tan W, Fan ZH, Qiu CX, Ricco AJ, Gibbons I.
    Journal: Electrophoresis; 2002 Oct; 23(20):3638-45. PubMed ID: 12412135.
    Abstract:
    We report the demonstration of miniaturized capillary isoelectric focusing (CIEF) in plastic microfluidic devices. Conventional CIEF technique was adapted to the microfluidic devices to separate proteins and to detect protein-protein interactions. Both acidic and basic proteins with isoelectric points (pI) ranging from 5.4 to 11.0 were rapidly focused, mobilized, and detected in a 1.2 cm long channel (50 microm deep x 120 microm wide) with a total analysis time of 150 s. In a device with a focusing distance of 4.7 cm, the separation efficiency for a basic protein, lysozyme, was achieved as high as 1.5 x 10(5) plates, corresponding to 3.2 million plates per meter. We also experimentally confirmed that IEF resolution is essentially independent of focusing length when the applied voltage is kept the same and within a range that it does not cause Joule heating. Further, we demonstrated the use of miniaturized CIEF to study the interactions between two pairs of proteins, immunoglobulin G (IgG) with protein G and anti-six histidine (anti-6xHis) with 6xHis-tagged green fluorescent protein (GFP). Using this approach, protein-protein interactions can be detected for as little as 50 fmol of protein. We believe miniaturized CIEF is useful for studying protein-protein interactions when there is a difference in pI between a protein-protein complex and its constitutent proteins.
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