Capillary isoelectric focusing of proteins and microorganisms in dynamically modified fused silica with UV detection

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2006-09, Vol.841 (1), p.152-159
• Main Authors: HORKA, Marie, RUZICKA, Filip, HORKY, Jaroslav, HOLA, Veronika, SLAIS, Karel
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Bacteria - isolation & purification; Biological and medical sciences; Candida - isolation & purification; CIEF; Dynamically modified FS; Enterococcus faecalis - isolation & purification; Escherichia coli - isolation & purification; Fundamental and applied biological sciences. Psychology; Fungi - isolation & purification; General pharmacology; Hydrogen-Ion Concentration; Isoelectric Focusing - instrumentation; Isoelectric Focusing - methods; Medical sciences; p I markers; Pharmacology. Drug treatments; Polyethylene Glycols - chemistry; Proteins - analysis; Proteins - isolation & purification; Proteins and microorganisms; Reproducibility of Results; Saccharomyces cerevisiae - isolation & purification; Silicon Dioxide - chemistry; Spacers; Spectrophotometry, Ultraviolet - methods; Staphylococcus aureus - isolation & purification; Staphylococcus epidermidis - isolation & purification; Stenotrophomonas maltophilia - isolation & purification; Streptococcus agalactiae - isolation & purification; UV detection
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2006.05.013

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 p I 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 5 × 10 2 to 1 × 10 3 with on-column UV detection at 280 nm.