Stanley Brian A, Neverova Irina, Brown Heather A, Van Eyk Jennifer E
Department of Physiology, Queen's University, Kingston, ON, Canada.
Proteomics. 2003 Jun;3(6):815-20. doi: 10.1002/pmic.200300388.
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.
为了使二维凝胶电泳（2-DE）观察到的心肌蛋白质组最大化，研究了以下两种情况的效果：（1）在组织匀浆过程中使用离子型或不同的两性离子去污剂；（2）将等电聚焦的“标准”去污剂3-[(3-胆酰胺丙基)二甲基氨基]-1-丙烷磺酸盐（CHAPS）改为两性离子去污剂氨基磺基甜菜碱-14（ASB-14）或N-癸基-N-N'-二甲基-3-铵基-1-丙烷磺酸盐（SB3-10）。与去污剂ASB-14、SB3-10或CHAPS相比，十二烷基硫酸钠被证明是心脏组织匀浆过程中提取蛋白质的更优去污剂。此外，对于二维凝胶的不同区域，ASB-14和SB3-10的提取效果均优于CHAPS。在大多数情况下，匀浆和聚焦条件的最佳组合并不涉及使用相同的去污剂。具体而言，发现能够混合匀浆和聚焦条件可以使人们在心脏组织的二维电泳分析中获得的蛋白质斑点分辨率和数量之间达到最佳平衡。经确定，用于心脏蛋白质常规检测的缓冲液的一个极佳初始组合是在含有ASB-14的缓冲液中进行初始匀浆，然后在含有CHAPS的缓冲液中进行聚焦。
