Horká Marie, Růzicka Filip, Horký Jaroslav, Holá Veronika, Slais Karel
Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veverí 97, 611 42 Brno, Czech Republic.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Sep 1;841(1-2):152-9. doi: 10.1016/j.jchromb.2006.05.013. Epub 2006 Jun 9.
We suggest a method for the reproducible and efficient capillary isoelectric focusing of proteins and microorganisms in the pH gradient 3-10. The method involves the segmental injection of the simple ampholytes, the solution of the selected electrolytes, and the sample mixture of bioanalytes and carrier ampholytes to the fused silica capillaries dynamically modified by poly(ethylene glycol), PEG 4000, which is added to the catholyte, the anolyte and injected solutions. In order to receive the reproducible results, the capillaries were rinsed by the mixture of acetone/ethanol between analyses. For the tracing of the pH gradients the low-molecular-mass pI markers were used. The simple proteins and the mixed cultures of microorganisms, Saccharomyces cerevisiae CCM 8191, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Staphylococcus aureus, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224, Staphylococcus epidermidis CCM 4418 and Stenotrophomonas maltophilia, were focused and separated by the method suggested. The minimum detectable number of microbial cells was 5x10(2) to 1x10(3) with on-column UV detection at 280 nm.
我们提出了一种在pH梯度为3 - 10的条件下，对蛋白质和微生物进行可重复且高效的毛细管等电聚焦方法。该方法包括将简单两性电解质、选定电解质溶液以及生物分析物与载体两性电解质的样品混合物分段注入到由聚乙二醇（PEG 4000）动态修饰的熔融石英毛细管中，PEG 4000添加到阴极电解液、阳极电解液和注入溶液中。为了获得可重复的结果，在每次分析之间用丙酮/乙醇混合物冲洗毛细管。使用低分子量pI标记物来追踪pH梯度。通过所建议的方法对简单蛋白质以及微生物混合培养物进行了聚焦和分离，这些微生物包括酿酒酵母CCM 8191、大肠杆菌CCM 3954、白色念珠菌CCM 8180、近平滑念珠菌、克鲁斯念珠菌、金黄色葡萄球菌、无乳链球菌CCM 6187、粪肠球菌CCM 4224、表皮葡萄球菌CCM 4418和嗜麦芽窄食单胞菌。在280 nm处进行柱上紫外检测时，微生物细胞的最低可检测数量为5×10²至1×10³ 。
