title: Multistage isoelectric focusing in a polymeric microfluidic chip.
abstract: This paper reports a protocol that improves the resolving power of isoelectric focusing (IEF) in a polymeric microfluidic chip. This method couples several stages of IEF in series by first focusing proteins in a straight channel using broad-range ampholytes and then refocusing segments of the first channel into secondary channels that branch from the first one at T-junctions. Experiments demonstrate that several fluorescent proteins that had focused within a segment of the straight channel in the first stage were refocused at significantly higher resolution due to the shallower pH gradient and higher electrical field gradient. Two variants of green fluorescent protein from the second-stage IEF fractionation were further separated in a third stage. Three stages of IEF were completed in less than 25 min at electric field strengths ranging from 50 to 214 V/cm.
authors.affiliation: Department of Chemical Engineering and School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164, USA.
authors.forename: Huanchun
authors.initials: H
authors.lastname: Cui
authors.forename: Keisuke
authors.initials: K
authors.lastname: Horiuchi
authors.forename: Prashanta
authors.initials: P
authors.lastname: Dutta
authors.forename: Cornelius F
authors.initials: CF
authors.lastname: Ivory
mesh_terms: D004129:Dimethylpolysiloxanes; D049452:Green Fluorescent Proteins / Q000302:isolation & purification; D007525:Isoelectric Focusing / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D011506:Proteins / Q000302:isolation & purification*; D012828:Silicones
chemical_list: D004129:Dimethylpolysiloxanes; D011506:Proteins; D012828:Silicones; D049452:Green Fluorescent Proteins; C013830:baysilon