BROWSE

Related Researcher

Author

Soper, Steven A.
Soper Research Group
Research Interests
  • Micro- and nano-fabrication

ITEM VIEW & DOWNLOAD

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity mu SPE device

Cited 2 times inthomson ciCited 0 times inthomson ci
Title
Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity mu SPE device
Author
Battle, Katrina N.Jackson, Joshua M.Witek, Małgorzata A.Hupert, Mateusz L.Hunsucker, Sally A.Armistead, Paul M.Soper, Steven A.
Keywords
PROTEOMIC ANALYSIS; PLASMA-MEMBRANES; MICROCHIP ELECTROPHORESIS; RELATIVE QUANTIFICATION; MASS-SPECTROMETRY; TITER PLATE; IN-VIVO; IDENTIFICATION; BIOTINYLATION; SEPARATION
Issue Date
201403
Publisher
ROYAL SOC CHEMISTRY
Citation
ANALYST, v.139, no.6, pp.1355 - 1363
Abstract
We present a novel microfluidic solid-phase extraction (μSPE) 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/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE 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 (∼20 fmol) of membrane proteins could be isolated and recovered with ∼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 μSPE device using computational simulations of different micropillar geometries to guide future device designs.
URI
Go to Link
DOI
http://dx.doi.org/10.1039/c3an02400h
ISSN
0003-2654
Appears in Collections:
SLS_Journal Papers

find_unist can give you direct access to the published full text of this article. (UNISTARs only)

Show full item record

qr_code

  • mendeley

    citeulike

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

MENU