title: Microchamber integration unifies distinct separation modes for two-dimensional electrophoresis.
abstract: By combining isoelectric focusing (IEF) with subsequent gel electrophoresis, two-dimensional electrophoresis (2DE) affords more specific characterization of proteins than each constituent unit separation. In a new approach to integrating the two assay dimensions in a microscope slide-sized glass device, we introduce microfluidic 2DE using photopatterned polyacrylamide (PA) gel elements housed in a millimeter-scale, 20-μm-deep chamber. The microchamber minimizes information loss inherent to channel network architectures commonly used for microfluidic 2DE. To define the IEF axis along a "lane" at the top of the chamber, we used free solution carrier ampholytes and immobilized acrylamido buffers in the PA gels. This approach yielded high-resolution (0.1 pH unit) and rapid (<20 min) IEF. Next, protein transfer to the second dimension was accomplished by chemical mobilization perpendicular to the IEF axis. Mobilization drove focused proteins off the IEF lane and into a region for protein gel electrophoresis. Using fluorescently labeled proteins, we observed transfer-induced band broadening factors ~7.5-fold lower than those observed in microchannel networks. Both native polyacrylamide gel electrophoresis (PAGE) and pore-limit electrophoresis (PLE) were studied as the second assay dimension and completed in <15 min. PLE yields protein molecular mass information without the need for ionic surfactant or reducing agents, simplifying device design and operation. Microchamber-based 2DE unifies two independent separation dimensions in a single device with minimal transfer-associated information losses. Peak capacities for the total assay ranged from 256 to 35 with <1 h assay duration. The rapid microchamber 2DE assay has the potential to bridge an existing gap in targeted proteomics for protein biomarker validation and systems biology that may complement recent innovation in mass spectrometry.
authors.affiliation: The UC Berkeley/UCSF Graduate Program in Bioengineering, Berkeley, California 94720, United States.
authors.forename: Augusto M
authors.initials: AM
authors.lastname: Tentori
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Hughes
authors.forename: Amy E
authors.initials: AE
authors.lastname: Herr
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004867:Equipment Design; D046210:Microfluidic Analytical Techniques; D011506:Proteins / Q000032:analysis*; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D011506:Proteins; D000099166:Polyacrylamides
references.citation: Lab Chip. 2007 Dec;7(12):1806-12
references.pmid: 18030404
references.citation: Anal Biochem. 1970 Jan;33(1):204-7
references.pmid: 5413240
references.citation: Anal Biochem. 1988 Dec;175(2):544-7
references.pmid: 3239780
references.citation: Lab Chip. 2008 Feb;8(2):316-22
references.pmid: 18231672
references.citation: J Am Chem Soc. 2012 Oct 24;134(42):17582-91
references.pmid: 23017083
references.citation: Anal Chem. 2008 May 1;80(9):3327-33
references.pmid: 18341355
references.citation: Anal Chem. 2011 May 1;83(9):3581-8
references.pmid: 21456518
references.citation: Anal Chem. 2009 Oct 1;81(19):8177-84
references.pmid: 19731927
references.citation: Anal Chem. 2012 Mar 6;84(5):2533-40
references.pmid: 22304398
references.citation: Anal Chem. 2010 May 1;82(9):3803-11
references.pmid: 20353191
references.citation: Annu Rev Biochem. 1998;67:509-44
references.pmid: 9759496
references.citation: Anal Chem. 2007 Oct 1;79(19):7485-91
references.pmid: 17718538
references.citation: Lab Chip. 2009 Sep 21;9(18):2729-37
references.pmid: 19704990
references.citation: Biochem Genet. 1979 Jun;17(5-6):499-516
references.pmid: 117795
references.citation: Anal Chem. 2003 Mar 1;75(5):1180-7
references.pmid: 12641239
references.citation: Electrophoresis. 2010 May;31(10):1606-14
references.pmid: 20419703
references.citation: Anal Chem. 2010 Feb 15;82(4):1253-60
references.pmid: 20092256
references.citation: Ann N Y Acad Sci. 1973 Jun 15;209:80-93
references.pmid: 4515055
references.citation: Nat Protoc. 2010 Nov;5(11):1844-56
references.pmid: 21030959
references.citation: Cancer Immunol Immunother. 2000 Nov;49(9):476-84
references.pmid: 11092614
references.citation: J Biol Chem. 1975 May 25;250(10):4007-21
references.pmid: 236308
references.citation: Nature. 1967 Jun 24;214(5095):1334-6
references.pmid: 6056848
references.citation: Clin Chem. 2004 Dec;50(12):2292-301
references.pmid: 15472031
references.citation: Metabolism. 1964 Oct;13:SUPPL:985-1002
references.pmid: 14228777
references.citation: J Chromatogr. 1977 Feb 21;132(3):451-68
references.pmid: 885944
references.citation: J Chromatogr A. 2002 Jul 12;962(1-2):21-7
references.pmid: 12198965
references.citation: Anal Chem. 1984 Oct;56(12):1258A-1260A, 1262A, 1264A passim
references.pmid: 6507850
references.citation: FEBS Lett. 1972 Feb 1;20(2):199-202
references.pmid: 11946415
references.citation: Lab Chip. 2009 Feb 21;9(4):592-9
references.pmid: 19190795
references.citation: J Am Chem Soc. 2010 Mar 3;132(8):2512-3
references.pmid: 20131779
references.citation: Lab Chip. 2008 Aug;8(8):1273-9
references.pmid: 18651068
references.citation: Anal Chem. 2009 Jan 15;81(2):845-50
references.pmid: 19053226
references.citation: Anal Chem. 2008 Jan 15;80(2):461-73
references.pmid: 18076145
references.citation: Proc Natl Acad Sci U S A. 1970 Apr;65(4):970-7
references.pmid: 4191703
references.citation: Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):5972-7
references.pmid: 22474344
references.citation: Nature. 1969 Mar 15;221(5185):1056-7
references.pmid: 5774398
references.citation: Anal Chem. 2010 Apr 15;82(8):3343-51
references.pmid: 20334346
references.citation: Lab Chip. 2010 Aug 21;10(16):2122-9
references.pmid: 20556309
references.citation: J Biochem Biophys Methods. 1984 May;9(2):103-19
references.pmid: 6736557