An efficient solubilization buffer for plant proteins focused in immobilized pH gradients.
The solubilization of a large array of proteins before electrophoresis itself is a very critical point for proteomic analyses. We compared the efficiency of several different solubilization buffers. From this work, we defined a very efficient solubilization buffer, including two chaotropes, two reducing agents (R2), two detergents (D2), and two kinds of carrier ampholytes in combination. This so-called R2D2 buffer (5 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, 2% N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate, 20 mM dithiothreitol, 5 mM Tris(2-carboxyethyl) phosphine, 0.5% carrier ampholytes 4-6.5, 0.25% carrier ampholytes 3-10) proved to be very efficient for a large range of different samples and allowed us to obtain two-dimensional gels of high resolution and quality.
Méchin
Valérie
V
INRA / INA-PG / UPS / CNRS UMR8120, Station de Génétique Végétale, Ferme du Moulon, 91190 Gif-sur-Yvette, France. mechin@moulon.inra.fr
Consoli
Luciano
L
Le Guilloux
Martine
M
Damerval
Catherine
C
D002021:Buffers; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods; D010940:Plant Proteins / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D018892:Proton-Motive Force; D019163:Reducing Agents / Q000494:pharmacology; D016622:Silver Staining
D002021:Buffers; D003902:Detergents; D010940:Plant Proteins; D011506:Proteins; D019163:Reducing Agents