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PubMed1996

Examination of capillary zone electrophoresis, capillary isoelectric focusing and sodium dodecyl sulfate capillary electrophoresis for the analysis of recombinant tissue plasminogen activator.

The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of omega-amino acid buffers (pH approximately 5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an epsilon-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 microns I.D. capillary or narrow bore capillaries without a polymer solution (25 microns I.D.) were employed. rtPA was resolved into at least eight species within a pI range of 6.4-9.2 using Ampholine 3.5-10. Migration time precision for the major peaks ranged from 0.2% for CZE to < or = 2-3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.

Acetates↗

PubMed2002

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) were used to identify iron-responsive proteins in the white-rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron-sufficient and iron-deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS-PAGE for P. chrysosporium and twelve for L. edodes. In 2-DE, the numbers of iron-restricted induced proteins were 12 and 9, respectively, in a resolution range of 15-60 kDa and pI 4.5-8.1. SDS-PAGE for the plasma membrane protein did not show differences, whereas the outer-membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron-complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.

Chrysosporium↗

PubMed1988

High resolution two-dimensional electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cheese proteins after rapid solubilisation.

The proteins of cheese are rapidly solubilised by heating to 95 degrees C in buffered 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol. Electrophoretic analysis of the solubilised proteins by either one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis or high resolution two-dimensional electrophoresis yields reproducible patterns characteristic of an individual cheese and its extent of ripening. The patterns reveal (i) the residual amounts of milk casein and whey proteins, and (ii) the appearance of casein degradation products, including pink-violet components as detected by Coomassie Blue staining.

Cheese↗

PubMedDate not supplied

Pulsed field gel electrophoresis: studies of DNA migration made with the programmable, autonomously-controlled electrode electrophoresis system.

We have studied the migration of DNA in pulsed field agarose gels under a variety of electrophoresis conditions. We have made use of an instrument which can generate electric fields of any orientation, magnitude, or duration to compare different separation techniques for DNA molecules of from 1 to several thousand kilobase pairs. We discuss the capabilities of the system and present results of gel runs in which electrophoresis conditions were changed individually or in combination. The mobility of DNA in pulsed field gels is shown to reflect a number of interdependent physical parameters.

Autoanalysis↗

PubMed1998

Ultrathin-layer zone electrophoresis of alcohol dehydrogenase in partly rehydrated polyacrylamide gels: an alternative to starch gel electrophoresis.

A highly sensitive electrophoretic technique for the separation of alcohol dehydrogenase isoenzymes by zone electrophoresis in partly rehydrated polyacrylamide gels is described. Five hundred microm thin polyacrylamide gels are polymerized under standardized conditions. After polymerization the gels are washed thoroughly with distilled water to remove any unreacted monomers, catalysts or still soluble polymers. The washed gels are then impregnated with 0.5% Tween 20 and dried. Before electrophoresis the dry gels are rehydrated to a thickness of 250 microm, which makes up 50% of the original gel volume. Rehydration is carried out by use of a degassed buffer solution. This method permits the demonstration of the isoenzymes of alcohol-dehydrogenase class I and II in man and allows quantitative determination.

Acrylic Resins↗

PubMed2004

Polymer solutions and entropic-based systems for double-stranded DNA capillary electrophoresis and microchip electrophoresis.

We give an overview of recent development of low-viscosity polymer solutions and entropic trapping networks for double-stranded DNA (dsDNA) separations by conventional capillary electrophoresis and microchip electrophoresis. Theoretical models for describing separation mechanisms, commonly used noncross-linked polymer solutions, thermoresponsive (viscosity-adjustable) polymer solutions, and novel entropic trapping networks are included. The thermoresponsive polymer solutions can be loaded at one temperature into microchannels at lower viscosities, and used in separation at another temperature at entanglement threshold concentrations and higher viscosities. The entropic-based separations use only arrays of regular obstacles acting as size-separations and do not need viscous polymer solutions. These progresses have potential in integration to automated capillary and microfluidic chip systems, enabling better reusability of separation microchannels, much shorter DNA separation times, and higher reproducibility due to less matrix degradation.

Electrophoresis, Capillary↗

PubMed2005

Nonradioactive telomerase activity assay by microchip electrophoresis: privileges to the classical gel electrophoresis assay.

The present study accents on the privileges of microchip-based electrophoresis to the conventional gel electrophoresis in separation of telomerase repeat amplification protocol/polymerase chain reaction (PCR) ladder products obtained in telomerase-catalyzed reaction in cancer cells. We try to clarify the interpretation of the results obtained by both electrophoretic procedures and to avoid misinterpretation as a result of PCR-dependent artefacts.

Electrophoresis, Microchip↗

PubMed2005

Analysis of poly(amidoamine)-succinamic acid dendrimers by slab-gel electrophoresis and capillary zone electrophoresis.

Ethylenediamine (EDA)-core poly(amidoamine) (PAMAM) succinamic acid dendrimers (Ex.SAH, where x refers to the generation) were synthesized and analyzed by polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), potentiometric acid-base titration, and capillary zone electrophoresis (CZE). Various generations (E1.SAH-E7.SAH) PAMAMs and a succinamic acid terminated core-shell tecto(dendrimer) (E5(E3.SAH)(n)) were first analyzed by PAGE. PAGE results show that the relative mobilities of generation 2 to generation 7 dendrimers decreased with the increasing number of generations. The molecular mass of a generation 5 core generation 3 shell tecto(dendrimer) (denoted as E5(E3.SAH)(n)) was determined to be between the Mw of E6.SAH and E7.SAH. CZE analysis allowed the evaluation of electrophoretic properties of given-generation dendrimers. The electrophoretic mobilities of individual generations PAMAM polyanions are similar, indicating that the separation mainly depends on their approximately identical charge/mass ratio. The E5(E3.SAH)(n) tectodendrimer had a lower electrophoretic mobility, which was consistent with its lower charge/mass ratio. The combination of PAGE and CZE analysis provides an alternative and effective way to characterize this group of PAMAM-succinamic acid dendrimers.

Dendrimers↗

PubMed2005

Determination of vitamin C and preservatives in beverages by conventional capillary electrophoresis and microchip electrophoresis with capacitively coupled contactless conductivity detection.

The separation and detection of commonly used preservatives (benzoate, sorbate) and vitamin C by both conventional CE and microchip electrophoresis with capacitively coupled contactless conductivity detection is presented. The separation was optimized by adjusting the pH-value of the buffer and the use of hydroxypropyl-beta-CD (HP-beta-CD) and CTAB as additives. For conventional CE, optimal separation conditions were achieved in a histidine/tartrate buffer at pH 6.5, containing 0.025% HP-beta-CD and 0.1 mM CTAB. LOD ranged from 0.5 to 3 mg/L (S/N = 3) and the RSDs for migration time and peak area were less than 0.1 and 2%, respectively. A considerable reduction of analysis time can be accomplished by using microchip electrophoresis without significant loss in sensitivity under optimal separation conditions. A histidine/tartrate buffer at pH 6.5, incorporating 0.06% HP-beta-CD and 0.25 mM CTAB, gave detection limits ranging between 3 and 10 mg/L and satisfactory reproducibilities of < or =0.4% for the migration time and < or =3.5% for the peak area. The methods developed are useful for the quantitative determination of food additives in real samples such as soft drinks and vitamin C tablets.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2006

Rapid analysis of atorvastatin calcium using capillary electrophoresis and microchip electrophoresis.

In this work, a capillary electrophoretic method for the rapid quantitation of atorvastatin (AT) in a lipitor tablet was investigated and developed. Method development included studies of the effect of applied potential, buffer concentration, buffer pH, and hydrodynamic injection time on the electrophoretic separation. The method was validated with regard to linearity, precision, specificity, LOD, and LOQ. The optimum electrophoretic separation conditions were 25 mM sodium acetate buffer at pH 6, with a separation voltage of 25 kV using a 50 microm capillary of 33 cm total length. Sodium diclofenac was used as an internal standard. Analysis of AT in a commercial lipitor tablet by electrophoresis gave quite high efficiency, coupled with an analysis time of less than 1.2 min in comparison to LC. Once the separation was optimized on capillary, it was further miniaturized to a microchip platform, with linear imaging UV detection using microchip electrophoresis (MCE). Linear imaging UV detection allowed for real-time monitoring of the analyte movement on chip, so that the optimum separation time could be easily determined. This microchip electrophoretic method was compared to the CE method with regard to speed, efficiency, precision, and LOD. This work represents the most rapid and first reported analysis of AT using MCE.

Atorvastatin↗

PubMed1997

Pulsed-field gel electrophoresis is more discriminating than multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis for typing pyogenic streptococci.

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S.dysgalactiae, 11 "S. equisimilis", three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci.

DNA, Bacterial↗

PubMed1995

Differences between natural and recombinant interleukin-2 revealed by gel electrophoresis and capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) is shown to be useful for analysis of interleukin-2 (IL-2) in its native state under non-reducing conditions. The results obtained were compared with those from analysis of IL-2 by protein blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing and reducing conditions. In addition, resolution of the different glycosylated and non-glycosylated natural IL-2 species was achieved by HPCE. The HPCE electropherogram of native IL-2 could easily generate quantitative amounts of the different naturally occurring IL-2 species. For HPCE of IL-2 run times of less than 10 min are sufficient, and only extremely small amounts of IL-2 are needed. In this report, human IL-2 expressed in bacteria has been analysed by HPCE and the existence of two recombinant IL-2 forms was demonstrated.

Electrophoresis, Capillary↗

PubMed2005

An investigation of pectin methylesterification patterns by two independent methods: capillary electrophoresis and polysaccharide analysis using carbohydrate gel electrophoresis.

The analysis of partially methylesterified oligogalacturonides plays a key role both in the elucidation of the fine structure of the polysaccharide pectin and in the study of pectin-acting enzymes. Experimental methods performing the separation, detection and quantification of oligogalacturonides are, therefore, of crucial importance in the drive to understand structure-function relationships in pectin containing systems, both in vitro and in vivo. In this work standard samples of unesterified and partially methylesterifed galacturonides, and enzymatic digests of several pectin samples possessing distinct intramolecular patterns of methylesterification were studied using capillary electrophoresis (CE) and polysaccharide analysis using carbohydrate gel electrophoresis (PACE). In addition to yielding interesting information regarding the fine structures of the different pectic substrates digested, the study has been used as a vehicle in order to compare the two recently reported methods.

Electrophoresis↗

PubMed2006

Analysis of TP53 gene mutations in human lung cancer: comparison of capillary electrophoresis single strand conformation polymorphism assay with denaturing gradient gel electrophoresis and direct sequencing.

INTRODUCTION: Automated capillary electrophoresis single strand conformation polymorphism (CE-SSCP), denaturant gradient gel electrophoresis (DGGE), and direct sequencing were compared to investigate the benefits and sensitivity of each of the methods for detection of unknown TP53 mutations in human lung cancer. METHODS: Twenty previously analyzed DNA samples from lung tumors were examined under dummy laboratory codes for occurrence of mutations of the TP53 gene. RESULTS: Mutations were found in 17 samples; 15 (88%) of them were detected by DGGE, 16 (94%) by CE-SSCP and 12 (71%) by direct sequencing. One of the two mutations that remained undetected in DGGE was in fact outside the sequence area covered by DGGE screening, thus rendering DGGE and CE-SSCP equally efficient in mutation detection. Direct sequencing performed less well in finding mutations than the two other assays, as also shown previously. CONCLUSION: The study showed that CE-SSCP is a fast and highly reproducible method, which is considerably less laborious compared to DGGE, for screening of unknown TP53 mutations.

DNA Mutational Analysis↗

PubMed2003

Capillary zone electrophoresis of soil humic acid fractions obtained by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis.

Capillary zone electrophoresis (CZE) was used for characterisation of soil humic acid (HA) fractions obtained by coupling size-exclusion chromatography with polyacrylamide gel electrophoresis, on the basis of their molecular size and electrophoretic mobility. CZE was conducted using several low alkaline buffers as background electrolyte (BGE): 50 mM carbonate, pH 9.0; 50 mM phosphate, pH 8.5; 50 mM borate, pH 8.3; 50 mM Tris-borate+1 mM EDTA+7 M urea+0.1% sodium dodecyl sulphate (SDS), pH 8.3. Independently of BGE conditions, the effective electrophoretic mobility of HA fractions were in good agreement with their molecular size. The better resolution of HA were obtained in Tris-borate-EDTA buffer with urea and SDS. This results indicated that CZE, mostly with BGE-contained disaggregating agents, is useful for separating HAs in fractions with different molecular sizes.

Chromatography, Gel↗

PubMed1998

Comparison of slab gel electrophoresis and capillary electrophoresis for the detection of the fluorescently labeled polymerase chain reaction products of short tandem repeat fragments.

The sizing capability of slab gel electrophoresis for short tandem repeat (STR) fragments was compared to the sizing capability of capillary electrophoresis (CE). Both systems used automated laser fluorescence detection to detect four fluorescent dyes, enabling the use of an internal lane standard within each sample. The STR fragments were amplified using a multiplex polymerase chain reaction (PCR) in which the STR fragments Hum CD-4, Hum TH01, Hum D21S11 and Hum SE33 were amplified simultaneously. The reproducibility of the size calling was determined for both systems. The average standard deviation obtained for the slab gel system was 0.2, which was comparable to the standard deviation of 0.12 obtained for the CE system. The CE system produced results comparable to those obtained on the slab gel system, with a level of precision of +/- 1.0 bp (between instruments).

DNA↗

PubMed1998

Use of capillary electrophoresis as a method development tool for classical gel electrophoresis.

Capillary electrophoresis (CE) was used to optimize the buffer pH, ionic strength and sulfated cyclodextrin concentrations for enantiomeric separation of piperoxan. These enantioseparation conditions were then applied to a classical gel electrophoresis system. Binding constants of the sulfated beta-cyclodextrin-piperoxan couple were approximated using CE and the effects of organic solvents on the system were also investigated.

Buffers↗

PubMed2000

Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.

Polymerase chain reaction (PCR) technique is widely used in the diagnosis of lymphoma, and PCR amplification products are typically detected by polyacrylamide gel electrophoresis (PAGE). However, the identification of small clonal populations, or the distinction of clonal PCR products in a polyclonal milieu remains difficult, requiring technically demanding alterations to gel analysis. This study describes an alternative approach using a capillary electrophoresis (CE) system to produce an accurately sized electropherogram. A variety of patient samples were examined, including solid tissue, peripheral blood, bone marrow aspirates, and paraffin-embedded tissue. A total of 28 samples were evaluated by PCR for B-cell clonality by detection of immunoglobulin heavy chain gene rearrangement and 29 samples for T-cell clonality by detection of T-cell gamma locus gene rearrangement. Standard 10% PAGE analysis of PCR products was compared with CE. There was a 100% concordance in the assessment of both B-cell and T-cell clonality. Dilution studies with the SUP-B15 cell line showed a detection limit of 0.03% for B-cell clonality and 0.05% for T-cell clonality using CE, versus 0.2% to 1%, respectively for PAGE. Automated, fluorescent analysis of PCR products by CE seems to be at least equally as effective as gel-based analysis for the detection of clonal B-cell and T-cell populations. Moreover. CE offers superior resolution and improved sensitivity, thus representing a significant improvement over traditional gel electrophoretic techniques in these regards.

B-Lymphocytes↗