title: Miniaturized capillary isoelectric focusing in plastic microfluidic devices.
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.
authors.affiliation: ACLARA BioSciences, 1288 Pear Avenue, Mountain View, CA 94043, USA.
authors.forename: Woei
authors.initials: W
authors.lastname: Tan
authors.forename: Z Hugh
authors.initials: ZH
authors.lastname: Fan
authors.forename: Charmaine X
authors.initials: CX
authors.lastname: Qiu
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Ricco
authors.forename: Ian
authors.initials: I
authors.lastname: Gibbons
mesh_terms: D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D049452:Green Fluorescent Proteins; D007074:Immunoglobulin G / Q000302:isolation & purification; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D008164:Luminescent Proteins / Q000302:isolation & purification; D008904:Miniaturization; D009419:Nerve Tissue Proteins / Q000302:isolation & purification; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification*
chemical_list: C029152:G-substrate; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins