Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation
Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, r...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2012
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| Online Access: | http://hdl.handle.net/1721.1/75011 https://orcid.org/0000-0002-9547-3251 https://orcid.org/0000-0001-7192-580X |
| _version_ | 1810998721151762432 |
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| author | Wen, Jian Wilker, Erik W. Yaffe, Michael B Jensen, Klavs F |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Wen, Jian Wilker, Erik W. Yaffe, Michael B Jensen, Klavs F |
| author_sort | Wen, Jian |
| collection | MIT |
| description | Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly(vinyl alcohol) (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370 V/cm), efficient cooling maintains solution temperature inside the separation channel controllably in the range 2−25 °C. Protein samples with a dynamic concentration range from μg/mL to mg/mL can be loaded into the microdevice at a flow rate of 1 mL/h and residence time of 12 min. By using a protein complex of nine proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device-to-device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Postdevice sample concentrations result in a 10−20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The microdevice has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches. |
| first_indexed | 2024-09-23T14:49:27Z |
| format | Article |
| id | mit-1721.1/75011 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:49:27Z |
| publishDate | 2012 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/750112022-10-01T22:40:48Z Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation Wen, Jian Wilker, Erik W. Yaffe, Michael B Jensen, Klavs F Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemical Engineering Koch Institute for Integrative Cancer Research at MIT Wen, Jian Wilker, Erik W. Yaffe, Michael B. Jensen, Klavs F. Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly(vinyl alcohol) (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370 V/cm), efficient cooling maintains solution temperature inside the separation channel controllably in the range 2−25 °C. Protein samples with a dynamic concentration range from μg/mL to mg/mL can be loaded into the microdevice at a flow rate of 1 mL/h and residence time of 12 min. By using a protein complex of nine proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device-to-device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Postdevice sample concentrations result in a 10−20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The microdevice has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches. United States. Army Research Office (Contract W911NF-07-D-0004) National Institutes of Health (U.S.) (Grant GM60594) National Institutes of Health (U.S.) (Grant GM68762) 2012-11-26T17:43:54Z 2012-11-26T17:43:54Z 2010-01 2009-09 Article http://purl.org/eprint/type/JournalArticle 0003-2700 1520-6882 http://hdl.handle.net/1721.1/75011 Wen, Jian et al. “Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation.” Analytical Chemistry 82.4 (2010): 1253–1260. https://orcid.org/0000-0002-9547-3251 https://orcid.org/0000-0001-7192-580X en_US http://dx.doi.org/10.1021/ac902157e Analytical Chemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) PMC |
| spellingShingle | Wen, Jian Wilker, Erik W. Yaffe, Michael B Jensen, Klavs F Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation |
| title | Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation |
| title_full | Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation |
| title_fullStr | Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation |
| title_full_unstemmed | Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation |
| title_short | Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation |
| title_sort | microfluidic preparative free flow isoelectric focusing system optimization for protein complex separation |
| url | http://hdl.handle.net/1721.1/75011 https://orcid.org/0000-0002-9547-3251 https://orcid.org/0000-0001-7192-580X |
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