Native DIGE of fluorescent plant protein complexes.
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 combination, sizes of complexes as well as their subunit composition can be compared after mixing samples from different physiological states. Plants interact specifically 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 fluorescent scanning at the Cy5 excitation wavelength. This property 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 fluorescence from endogenous bound chlorophyll for identification of pigment-binding complexes and complex subunits.
Center of Organelle Research (CORE), University of Stavanger, Stavanger, Norway.
Veronika
V
Reisinger
Lutz Andreas
LA
Eichacker
D053000:Analytic Sample Preparation Methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods*
D005456:Fluorescent Dyes; D010940:Plant Proteins