Biology Reference
In-Depth Information
Chapter 46
Separation of Membrane Protein Complexes
by Native LDS-PAGE
Janine Arnold , Alexey Shapiguzov , Geoffrey Fucile , Jean-David Rochaix ,
Michel Goldschmidt-Clermont , and Lutz Andreas Eichacker
Abstract
Gel electrophoresis has become one of the most important methods for the analysis of proteins and protein
complexes in a molecular weight range of 1-10
7
kDa. The separation of membrane protein complexes
remained challenging to standardize until the demonstration of Blue Native PAGE in 1991 [1] and Clear
Native PAGE in 1994 [2]. We present a robust protocol for high-resolution separation of photosynthetic
complexes from
Arabidopsis thaliana
using lithium dodecyl sulfate as anion in a modifi ed Blue Native
PAGE (LDS-PAGE). Here, non-covalently bound chlorophyll is used as a sensitive probe to characterize
the assembly/biogenesis of the pigment-protein complexes essential for photosynthesis. The high fl uores-
cence yield recorded from chlorophyll-binding protein complexes can also be used to establish the separa-
tion of native protein complexes as an electrophoretic standard.
Key words
Native LDS-PAGE, Thylakoid membrane, Chlorophyll-binding protein complexes,
Fluorescence
Abbreviations
APS
Ammonium persulfate
DDM
n
-Dodecyl-
β
-
D
-maltoside
DIG Digitonin
LDS Lithium dodecyl sulfate
PAGE Polyacrylamide electrophoresis
TEMED Tetramethylethylenediamin
1
Introduction
Assemblies of proteins constitute molecular machines that make up
the functional proteome of a cell [
3
]. These assemblies can be
viewed as a highly dynamic network of protein interactions that
refl ect the cell's physiological state. It is therefore no wonder that
