Native DIGE of Fluorescent Plant Protein Complexes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 343

Chapter 24
Native DIGE of Fluorescent Plant Protein Complexes
Veronika Reisinger and Lutz Andreas Eichacker
Abstract
CyDye labeling and DIGE have not only been proven to work for soluble proteins but also at the level of
whole membrane protein complexes. After complex solubilization and CyDye labeling, proteins can be
separated by native PAGE which is often combined with SDS PAGE in a subsequent step. By this combina-
tion, sizes of complexes as well as their subunit composition can be compared after mixing samples from
different physiological states. Plants interact specifi cally with light via protein-bound pigments. This can be
used in combination with CyDye technology to extend the “classical” approach in plant research. As an
example, chlorophyll can be excited for fl uorescent scanning at the Cy5 excitation wavelength. This prop-
erty can be used to identify pigment-binding plant complexes and complex subunits isolated from plastid
membranes. In this protocol, we present a combination of the conventional CyDye labeling technique
with 2D native/SDS PAGE and parallel scanning for CyDyes and fl uorescence from endogenous bound
chlorophyll for identifi cation of pigment-binding complexes and complex subunits.
Key words: Native PAGE, DIGE, Membrane protein complexes, Chlorophyll fl uorescence,
Chlorophyll-binding proteins, Plant, Membrane, Protein complexes, Chlorophyll, CyDye
1. Introduction
Two-dimensional (2D) difference gel electrophoresis (DIGE) is a
powerful tool to quantify proteomes isolated from different physi-
ological states. Originally, this technique was invented for the overall
hydrophilic water-soluble part of a proteome in combination with
separation of proteins by 2D IEF/SDS PAGE ( 1 ). But different
publications have proven the suitability of CyDye labeling for the
analysis of membrane protein complexes as well ( 2-5 ). As IEF has
been shown to work suboptimal with membrane proteins ( 6 ), CyDye
labeling of membrane proteins is often combined with native PAGE.
There are different protocols for native PAGE separations available
( 7-10 ), among which blue native (BN) PAGE represents the most