Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium.
In order to maximize the myocardial proteome observed by two-dimensional gel electrophoresis (2-DE), the effect of (1) either an ionic or different zwitterionic detergents during tissue homogenization and (2) altering the "standard" detergent for isoelectric focusing (3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS)) to either the zwitterionic detergent amidosulfobetaine-14 (ASB-14) or N-decyl-N-N'-dimethyl-3-ammonio-1-propane sulfonate (SB3-10) was investigated. Sodium dodecyl sulfate was shown to be a superior detergent for extraction of proteins during homogenization of cardiac tissue compared to the detergents ASB-14, SB3-10 or CHAPS. Additionally, both ASB-14 and SB3-10 exhibited better extraction than CHAPS for distinct regions of two-dimensional gels. In most cases, the best combination of homogenization and focusing conditions did not involve the use of the same detergent. Specifically, it was found that the ability to mix homogenization and focusing conditions can allow one to obtain an optimum balance between the resolution and number of protein spots obtained in 2-DE analysis of cardiac tissue. An excellent initial combination of buffers to utilize for the general examination of cardiac proteins was determined to be initial homogenization in a buffer containing ASB-14 followed by focusing in a buffer containing CHAPS.
Stanley
Brian A
BA
Department of Physiology, Queen's University, Kingston, ON, Canada.
Neverova
Irina
I
Brown
Heather A
HA
Van Eyk
Jennifer E
JE
D002021:Buffers; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006352:Heart Ventricles; D006801:Humans; D007525:Isoelectric Focusing; D009124:Muscle Proteins / Q000302:isolation & purification*; D009206:Myocardium / Q000737:chemistry*; D020543:Proteome / Q000302:isolation & purification*; D012995:Solubility; D014020:Tissue Extracts / Q000302:isolation & purification
D002021:Buffers; D003902:Detergents; D009124:Muscle Proteins; D020543:Proteome; D014020:Tissue Extracts