Miniaturized capillary isoelectric focusing in plastic microfluidic devices.
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.
Tan
Woei
W
ACLARA BioSciences, 1288 Pear Avenue, Mountain View, CA 94043, USA.
Fan
Z Hugh
ZH
Qiu
Charmaine X
CX
Ricco
Antonio J
AJ
Gibbons
Ian
I
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*
C029152:G-substrate; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins