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Two-Dimensional 16-BAC/SDS Polyacrylamide Gel Electrophoresis of Mitochondrial Membrane Proteins

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Functional Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1871))

Abstract

The substitution of the reverse polarity benzyldimethyl-n-hexadecylammonium chloride (16-BAC) polyacrylamide gel electrophoresis (PAGE) for isoelectric focusing (IEF) in the first dimension of electrophoresis improves the solubility of extremely hydrophobic proteins and their recovery compared to conventional 2D IEF/SDS PAGE. The acidic environment of 16-BAC PAGE has also been shown to better preserve the labile methylation of basic proteins such as the histones. Several improvements of the 2D 16-BAC/SDS PAGE method are collectively described here with particular emphasis on the separation of mitochondrial membrane proteins of low molecular mass. Lowering the 16-BAC concentration 50-fold in the gel and buffers decreases the formation of mixed 16-BAC/SDS micelles, which otherwise interferes with the separation of very low molecular mass proteins in second dimension SDS PAGE, and consequently improved the resolution of mitochondrial membrane proteins in the 10–30 kDa range.

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Abbreviations

16-BAC:

Benzyldimethyl-n-hexadecylammonium chloride

CMC:

Critical micelle concentration

DTT:

Dithiothreitol

HED:

Hydroxyethyl disulfide

IEF:

Isoelectric focusing

KDS:

Potassium dodecylsulfate

PAGE:

Polyacrylamide gel electrophoresis

PMSF:

Phenylmethylsulfonyl fluoride

SDS:

Sodium dodecylsulfate

TCEP:

Tris (2-carboxyethyl) phosphine

TMDs:

Transmembrane domains

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Author information

Authors and Affiliations

  1. Focus Proteomics, Hudson, NH, USA

    Gary Smejkal & Srikanth Kakumanu

Authors
  1. Gary Smejkal
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  2. Srikanth Kakumanu
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Editor information

Editors and Affiliations

  1. Array Bridge Inc., St. Louis, MO, USA

    Xing Wang

  2. Biotech Support Group LLC, Monmouth Junction, NJ, USA

    Matthew Kuruc

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Smejkal, G., Kakumanu, S. (2019). Two-Dimensional 16-BAC/SDS Polyacrylamide Gel Electrophoresis of Mitochondrial Membrane Proteins. In: Wang, X., Kuruc, M. (eds) Functional Proteomics. Methods in Molecular Biology, vol 1871. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8814-3_3

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  • DOI: https://doi.org/10.1007/978-1-4939-8814-3_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8813-6

  • Online ISBN: 978-1-4939-8814-3

  • eBook Packages: Springer Protocols

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