Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii.
Proteins of haloarchaea are remarkably unstable in low-ionic-strength solvents and tend to aggregate under standard two-dimensional (2-D) gel electrophoresis conditions, causing strong horizontal streaking. We have developed a new approach to generate 2-D maps of halophilic proteins which included washing cells with 1.5 M Tris-HCl buffer. In addition, proteins were precipitated with acetone, solubilized with urea and thiourea in the presence of the sulfobetaine detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), reduced with tributylphosphine (TBP), and separated with microrange strips of immobilized pH gradients (pH 3.9-5.1). This combination enabled the construction of highly reproducible 2-D maps of Haloferax volcanii proteins.
Karadzic
Ivanka M
IM
Department of Microbiology and Cell Science, University of Florida, Gainsville, FL 32611, USA.
Maupin-Furlow
Julie A
JA
D000096:Acetone / Q000494:pharmacology; D001105:Archaea / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D019622:Haloferax volcanii / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010720:Phosphines / Q000494:pharmacology; D020543:Proteome; D012995:Solubility; D013890:Thiourea / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D002793:Cholic Acids; D003902:Detergents; D010720:Phosphines; D020543:Proteome; C043255:tri-n-butylphosphine; D000096:Acetone; D014508:Urea; D013890:Thiourea; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate