Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds.
Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4-6 and pH 3-10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6-11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots.
Chinnasamy
Gurusamy
G
Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9. gchinnas@lawsonimaging.ca
Rampitsch
Christof
C
D000665:Ampholyte Mixtures; D002021:Buffers; D002793:Cholic Acids; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods; D010940:Plant Proteins / Q000302:isolation & purification*; D019163:Reducing Agents; D012639:Seeds / Q000737:chemistry; D012995:Solubility; D013890:Thiourea; D014908:Triticum / Q000737:chemistry*; D014508:Urea
D000665:Ampholyte Mixtures; D002021:Buffers; D002793:Cholic Acids; D010940:Plant Proteins; D019163:Reducing Agents; D014508:Urea; D013890:Thiourea; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate