Separation of isoforms of Serratia marcescens nuclease by capillary electrophoresis

Pedersen, Jacob Krabbe; Pedersen, Mikael; Søeberg, H.; Biedermann, Kirsten

doi:10.1016/0021-9673(93)83395-9

Cited by 13 publications

(5 citation statements)

References 12 publications

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“…Capillary isoelectric focusing (CIEF) is one separation mode of CE, based on the difference in isoelectric point (pl) of amphoteric analytes [ll-121. The resolution of CIEF, in many cases, is superior to that of other modes of CE [13]. In the conventional CIEF procedure, first a pH gradient is formed in a capillary, and protein samples are focused at points where the pH is equal to the p l of the respective proteins.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence imaging detection for capillary isoelectric focusing

Pawliszyn

1995

Electrophoresis

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A simple laser-induced fluorescence (LIF) imaging detector and an ultrasensitive LIF imaging detector are described for capillary isoelectric focusing (CIEF). An argon ion laser beam of 496.5 nm is used as excitation source. In the simple LIF imaging detector, the excitation beam is directed into a capillary column by an optic fiber array. In the ultrasensitive LIF imaging detector, the laser beam is first expanded, then is focused into the 4.5 cm long capillary column by a cylindrical lens. Fluorescence emission is detected by a charge-coupled device (CCD) camera. The feasibility and performance of the LIF imaging detector system for CIEF are first verified with a naturally fluorescent protein, b-phycoerythrin. Then, the ultrasensitive LIF imaging system is used as a detector for CIEF of proteins labeled with fluorescein isothiocyanate (FITC). Three FITC-labeled proteins (i) alpha-D-galatosylated FITC-albumin, (ii) insulin-FITC, and (iii) casein-FITC, are used as model samples. Fluorescence images of the model samples are measured during the CIEF process. The focusing of the protein samples is complete in about 1.5 min. The ultrasensitive detector's detection limits for the FITC-labeled proteins are at the level of 10(-10) M, and the mass detection limits are about 4.5 x 10(-17) mole, even though only 10% of the fluorescence emission is collected. Therefore, the method is capable of separating and detecting 10(-11) M or amole (10(-18) mole) level protein samples with a band-pass filter more specific to the fluorescence light.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Introductionmentioning

confidence: 99%

Fluorescence imaging detection for capillary isoelectric focusing

Pawliszyn

1995

Electrophoresis

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“…This paper reexamines the dependence of plate number N on surfactant concentration in micellar electrokinetic chromatography (MEKC) for the common surfactant, sodium dodecyl sulfate (SDS). Because this subject has been examined to various degrees in previous studies, − this paper may seem redundant. However, as we shall show, some of these studies may have been subject to limitations on N due to effects other than surfactant concentration, and trends drawn from them may be misleading.…”

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confidence: 99%

Reexamination of Dependence of Plate Number on SDS Concentration in Micellar Electrokinetic Chromatography

Seals

Davis

1996

Anal. Chem.

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Chromatograms of hydrophilic, hydrophobic, and intermediate-polarity analytes were developed in 50-μm capillaries by micellar electrokinetic chromatography at field strengths less than 31 kV/m. The analytes were solubilized by phosphate/borate buffers containing 15, 50, and 100 mM sodium dodecyl sulfate (SDS). The plate numbers N of the analytes, as well as those of the electroosmotic flow and micellar markers, were compared to predictions of N estimated by a simple model based on longitudinal diffusion and plug size. Good to fair agreement between theory and experiment was obtained for the hydrophilic and intermediate-polarity analytes in all buffers over the entire field strength range. Good agreement between theory and experiment was obtained for the hydrophobic analyte and micellar marker in all buffers at low field strengths; however, these compounds were subject to dispersion at higher field strengths by what appears to be Joule heating. The magnitudes of other, closely related Joule heating losses are quantified here using temperature profile measurements by Morris and co-workers and Taylor dispersion calculations. In contrast to the commonly reported increase of N with media concentration, the Ns of the hydrophilic and intermediate-polarity analytes were found to be essentially independent of SDS concentration over the investigated SDS range, and the Ns of the hydrophobic species were found to be independent of SDS concentration until (what appears to be) Joule heating became significant. These results were compared to those of Sepaniak and Cole. A critique of some previous studies of N vs SDS concentration is presented, in which quantitative explanations for some dispersions are offered as alternatives to surfactant concentration effects.

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“…In addition to these major isoforms, other isoforms present only as minor species have been identified by capillary electrophoresis [42] and electrospray mass spectrometry [43–45]. These have further short deletions at the N‐terminus.…”

Section: Processing Of the Nuclease Precursormentioning

confidence: 99%

Serratia marcescensand its extracellular nuclease

Benedik

Strych

1998

FEMS Microbiology Letters

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Serratia marcescens produces an endonuclease with extraordinarily high specific activity that is released into the surrounding medium. This enzyme has been the focus of studies on gene regulation, protein secretion, endonuclease action, and protein structure; it has also been found to have many applications in biotechnology. Here we briefly review these different facets of research regarding the Serratia nuclease and summarize the current state of knowledge about this enzyme.

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Separation of isoforms of Serratia marcescens nuclease by capillary electrophoresis

Cited by 13 publications

References 12 publications

Fluorescence imaging detection for capillary isoelectric focusing

Fluorescence imaging detection for capillary isoelectric focusing

Reexamination of Dependence of Plate Number on SDS Concentration in Micellar Electrokinetic Chromatography

Serratia marcescensand its extracellular nuclease

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