期刊
ANALYST
卷 139, 期 6, 页码 1355-1363出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/c3an02400h
关键词
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资金
- University Cancer Research Fund (UCRF) of the University of North Carolina
- National Institutes of Health [R21CA173279]
- National Science Foundation
We present a novel microfluidic solid-phase extraction (mu SPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O-3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the mSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (similar to 20 fmol) of membrane proteins could be isolated and recovered with similar to 89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the mSPE device using computational simulations of different micropillar geometries to guide future device designs.
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