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

  • Gary Smejkal
  • Srikanth Kakumanu
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Benzyldimethyl-n-hexadecylammonium chloride Cationic detergents Membrane proteins Mitochondria Polyacrylamide gel electrophoresis Proteins Sodium dodecylsulfate Two-dimensional gel electrophoresis Transmembrane domains 

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Gary Smejkal
    • 1
  • Srikanth Kakumanu
    • 1
  1. 1.Focus ProteomicsHudsonUSA

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