Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis.
Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii).
Harder
A
A
Technical University of Munich, Department of Food Technology, Freising-Weihenstephan, Germany.
Wildgruber
R
R
Nawrocki
A
A
Fey
S J
SJ
Larsen
P M
PM
Görg
A
A
D002021:Buffers; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D012995:Solubility
D002021:Buffers; D005656:Fungal Proteins