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PubMed1995

Separation of r-hirudin from similar substances by capillary electrophoresis.

The thrombin inhibitor r-hirudin is a peptide of 65 amino acids and with a pI of 4.4. Capillary electrophoresis (CE) was used to separate r-hirudin from seven closely related substances which may be found as by-products or from degradation. Possibly two of these substances differ only in an isoaspartyl instead of an aspartyl binding. A baseline separation was possible with an acetate buffer (pH 4.4, 60 mM) containing 0.3% (m/m) PEG 20,000 and 0.1 mM Zn2+. The possibilities to prevent wall adsorption are discussed.

Acetates↗
PubMed1995

Analysis of internucleosomal DNA fragmentation in apoptotic thymocytes by dynamic sieving capillary electrophoresis.

The analysis, by slab gel electrophoresis, of internucleosomal DNA cleavage or laddering, characteristic of apoptosis in many cell systems, is labour intensive, difficult to automate and at best only semi-quantitative. In this report we show that CE, using dilute solutions of hydroxyethylcellulose as a replaceable sieving matrix, can be applied to the relatively rapid analysis of DNA laddering in whole digests of apoptotic rat thymocytes. Also, using the sensitivity of laser-induced fluorescence detection and the highly sensitive nuclei acid stain YO-PRO-1, the CE method reported here can use 1000-2000 fold fewer cells than needed for traditional slab gel methods.

Animals↗
PubMed1994

Molecular size determinations of DNA restriction fragments and polymerase chain reaction products using capillary gel electrophoresis.

Commercially available capillary electrophoresis (CE) systems offer advantages over traditional slab gel methodologies. The capillary format allows the use of higher voltages (225 V/cm), which results in faster migration, higher resolution and greater efficiency without excessive heating. The ability to automate the system increases the unattended sample analysis throughput. For this study, the CE system was configured with a muPAGE 3% T, 3% C polyacrylamide gel capillary with an effective length of 40 cm and microPAGE Tris-borate urea buffer system. The analysis of DNA restriction fragments and polymerase chain reaction products, with an internal standard of Boehringer Mannheim DNA marker XI were completed in less than 70 min. All samples were analyzed at 260 nm. The data establishes automated capillary gel electrophoresis as a high-resolution, reproducible method for analysis of samples under 1000 base pairs.

Base Composition↗
PubMed1994

Simultaneous high-performance capillary electrophoretic determination of reduced and oxidized glutathione in red blood cells in the femtomole range.

This paper describes a high-performance capillary electrophoretic (HPCE) method which allows a quick, simultaneous and quantitative determination of reduced (GSH) and oxidized (GSSG) glutathione in mammalian red blood cells using a Supelco-bonded hydrophilic phase capillary CElect-P150. The extraction procedure of GSH and GSSG from erythrocytes using Microcon-10 membranes is very simple and allows a correct evaluation of these compounds present in the red blood cells. Furthermore, the HPCE method does not require removal of the excess N-ethylmaleimide used to block the glutathione in its reduced state, making the simultaneous evaluation of GSH and GSSG possible in a very short time (ca. 4 min), with a sensitivity at femtomole level.

Animals↗
PubMed1994

Evaluation of a novel hydrophilic derivatized capillary for protein analysis by capillary electrophoresis-electrospray mass spectrometry.

A new type of hydrophilic derivatized capillary has been used to enable the on-line capillary electrophoresis separation and electrospray mass spectrometric detection of a mixture of proteins containing bovine cytochrome c, tuna cytochrome c and horse heart myoglobin. Less than 40 fmol of each compound were loaded into the capillary. Baseline resolution of components was achieved, as were accurate assignments of molecular masses. The hydrophilic derivatized capillaries were taken through extensive testing procedures to characterize their performance and capabilities for protein analysis. A mixture of six proteins (cytochrome c, ribonuclease A, alpha-chymotrypsinogen, myoglobin, carbonic anhydrase II and alpha-lactalbumin) in acetic acid-sodium acetate buffer was used to delineate the relationships between migration time and pH, along with migration time and buffer concentration for each protein. The variations in capillary efficiency as a function of pH and as a function of buffer concentration were also characterized for the same six proteins in the acetic acid-sodium acetate system. A pH of 4.8 was found to offer an excellent compromise between separation efficiency (up to 500,000 theoretical plates) and analysis time. Capillary efficiencies were also found to be very good when employing a Tris.HCl electrolyte adjusted to pH 4.8. Lastly, electropherogram reproducibility and capillary durability were examined with the finding that little deterioration of the capillary occurred over the course of 400 injections (200 h run time). This represents a notable improvement over previously documented derivatization procedures designed to reduce protein adsorption to fused-silica capillary walls.

Animals↗
PubMed1994

Control of the cultivation process of antithrombin III and its characterization by capillary electrophoresis.

The production by baby hamster kidney cells of recombinant antithrombin III (r-AT III), the main inhibitor of thrombin, factor Xa and other proteases of the clotting cascade, was monitored by capillary isotachophoresis using mixtures of continuous spacers. The results were compared with those obtained by capillary zone electrophoresis (CZE). The downstream process, which incorporated anion-exchange and heparin affinity chromatography, was monitored by CZE under acidic conditions and voltage ramping. The purified product was characterized by its isoelectric point and molecular mass. Isoelectric points of the three major and three minor isoforms of AT III were evaluated by capillary isoelectric focusing using a pH range of 4-6 and various mobilization procedures. The molecular mass of AT III was investigated by capillary gel electrophoresis (CGE), applying removable dextran gels. Both parameters could be determined within 30 min using only one coated capillary. The results showed an excellent correspondence with those achieved with conventional slab gels. The affinity complex between AT III and thrombin could also be detected by CGE and the heparin dependence of the affinity reaction could be investigated.

Animals↗
PubMed1994

Affinity capillary electrophoresis applied to the studies of interactions of a member of heat shock protein family with an immunosuppressant.

The bioaffinity of receptor-ligand interactions is investigated by determining the binding constant (association constant or dissociation constant) of the resulting complex utilizing affinity capillary electrophoresis (ACE). The ACE binding assay was established with a potent immunosuppressant, deoxyspergualin (DSG), that binds specifically to Hsc70, a constitutive or cognate member of heat shock protein 70 (Hsp70) family. Quantitative determination of binding constants under different running buffer systems provide comparative results. The association constants for the interaction between Hsc70 protein and DSG were found to be 5.7 x 10(4) M-1 in a buffer with pH 6.95 and 6.3 x 10(4) M-1 in a buffer with pH 5.30 (or corresponding dissociation constants, 18 and 16 microM, respectively) based on Scatchard analyses. Binding of DSG to a synthetic peptide, SINPDEAVAYGAAVQAAILSGDK, one of the DSG-binding fragments found from tryptic digest of Hsc70 protein, provides further detailed information for the understanding of Hsc70 binding domain. The applicability of using coated capillaries was also evaluated for probing Hsc70-DSG interaction.

Amino Acid Sequence↗
PubMed1994

Determination of antigen-antibody affinity by immunocapillary electrophoresis.

The use of affinity capillary electrophoresis for the characterization of antigen-antibody interactions (immunocapillary electrophoresis) is shown using monoclonal antibodies against phosphotyrosine as a model system. The influence of the interaction kinetics on the peak profiles was demonstrated in experiments with addition of phosphotyrosine to the electrophoresis buffer. One of the two antibodies that were tested exhibited peak broadening while the other showed no change in peak shape but had a decreased mobility proportional to the amount of phosphotyrosine present. The migration shifts which were of the order 0.05 to 0.15 min at 439 V/cm were a consequence of the antibody-antigen complexes having a slower mobility than the non-complexed antibody. On the basis of measurement of migration shifts at different antigen concentrations, dissociation constants were estimated and shown to be independent on the applied field strength. Thus, when certain requirements are fulfilled, immuno-capillary electrophoresis is a fast and simple method for establishing binding characteristics of unlabelled antigen and antibody molecules under non-denaturing conditions and consumes minute amounts of sample.

Antibodies, Monoclonal↗
PubMed1994

Characterization of digoxigenin-labeled B-phycoerythrin by capillary electrophoresis with laser-induced fluorescence. Application to homogeneous digoxin immunoassay.

A laser-induced fluorescence (LIF) immunoassay technique based on capillary electrophoretic (CE) separation is demonstrated. The analysis of digoxin in serum at clinically useful concentration levels of 10(-9) to 10(-10) M is achieved using this technique. The chemistry presented here using digoxigenin-labeled B-phycoerythrin was selected as a convenient model for the exploration of CE-LIF-based immunoassays. The LIF system described here exhibits detection limits in the low 10(-11) M range for several common fluorophores. The data presented in this report are one of the first examples of nanomolar quantitative analysis in a human serum matrix by CE.

Binding, Competitive↗
PubMed1994

High-performance capillary electrophoresis for characterization of hapten-protein conjugates used for production of antibodies against soyasaponin I.

Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten-protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea (Pisum sativum L.) and soybean [Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten (p-nitrophenyl-alpha-D-galactoside; PNPG) coupled to KSTI. The PNPG-KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten-protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.

Animals↗
PubMed1994

Capillary electrophoresis of the scrapie prion protein from sheep brain.

Scrapie in sheep and goats causes a progressive, degenerative disease of the central nervous system and is the prototype of other transmissible spongiform encephalopathies (TSE) found in humans and in animals. In samples of TSE-affected brains, unique rod-shaped structures are found and are infectious. These rods are composed of a protease-resistant, post-translationally modified cellular protein (PrPsc) that has a molecular mass of ca. 27,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laboratory tests used for the diagnosis of scrapie detect PrPsc. The overall concentration of PrPsc in tissues is low. The present methods to diagnose scrapie are lengthy, require relatively large quantities of starting material to detect PrPsc and lack sensitivity. We explored the use of free zone capillary electrophoresis and immunocomplex formation to detect PrPsc in the brain tissue of infected sheep. Brain tissue from both infected (as confirmed by histological and biological tests) and from normal animals was used to prepare the PrPsc. After treatment with proteinase K and non-ionic detergents, PrPsc was solubilized and reacted with a rabbit antiserum specific for a peptide of the prion protein. Immunocomplex formation was observed for the samples from scrapie-infected brain but not for samples from normal brain. When a fluorescein-labeled goat anti-rabbit immunoglobulin was used as a second antibody, the detection of immunocomplex formation was enhanced both by the immunological technique and by using laser-induced fluorescence for detection. This same rabbit antiserum was used on immunoblot analysis. Three bands were observed for material from an infected sheep but none in preparations from brain material from normal sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Analysis of nucleotide pools in human lymphoma cells by capillary electrophoresis.

Capillary electrophoresis is applied to determine nucleotide pool levels in human Burkitt lymphoma cells. The analysis was performed on a 65 cm x 50 microns I.D. Ucon-coated column with on-column UV detector. The method requires only nanoliters of sample and a simple sample preparation procedure. Over 12 nucleotides were separated and quantitated with high resolution and reproducibility. The whole capillary electrophoretic separation time was only 35 min. These results demonstrate that capillary electrophoresis provides a useful and easy way to analyze nucleotide pools in cells.

Adenine Nucleotides↗
PubMed1994

Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations.

Quantitative capillary gel electrophoresis (QCGE) has been developed for the accurate quantitation of a 21-mer phosphorothioate oligonucleotide, ISIS 2922, and its degradation products in an intravitreal formulation. The electrokinetic mode of injection employed by CGE necessitates formulation of the external reference standard in a sample matrix similar to that of the drug product and the use of an internal standard for improved accuracy and precision. The analytical method detailed in this paper has demonstrated the necessary accuracy, precision, linearity, range, selectivity and ruggedness for use in routine drug product analysis and stability monitoring of phosphorothioate oligonucleotides.

Capillary Action↗
PubMed1994

Capillary gel electrophoresis of oligonucleotides: prediction of migration times using base-specific migration coefficients.

Chemically synthesized oligodeoxyribonucleotides were subjected to capillary gel electrophoresis on three different polyacrylamide-based matrices. Analysis of about 1000 samples over a 1-year period showed that the gel matrix evolved with time resulting in shifting migration times, making it essential to use an internal standard. Cross-linked polyacrylamide matrices had the highest stability, allowing an average of 100 injections on the same capillary. Computer-aided prediction of migration times was subsequently evaluated to confirm the size and base composition of oligonucleotides more accurately. A number of problems were noted when using this approach on a routine basis, such as insufficient stability of the gel matrices, effects of secondary structure on migration and insufficient differences in migration times for oligonucleotides containing > 50 bases. Capillary gel electrophoresis at pH 3.5 in replaceable gels showed that migration was mainly dependent on the charge per base ratio resulting in separations of significantly altered selectivity which complemented analyses under the commonly used basic pH conditions.

Base Sequence↗
PubMed1994

High-speed and high-accuracy DNA sequencing by capillary gel electrophoresis in a simple, low cost instrument. Two-color peak-height encoded sequencing at 40 degrees C.

A low-cost DNA sequencer was constructed based on a single helium-neon laser. The two-color peak-height encoded sequencing protocol, based on the use of T7 DNA polymerase in a manganese buffer, was used to generate samples. Two termination reactions were performed. In the first, a TAMRA (applied Biosystems)-labeled primer was extended in the presence of ddATP and ddCTP. The amounts of dideoxynucleotides were adjusted to produce a 3:1 peak height ratio. Similarly, a ROX (Applied Biosystems)-labeled primer was extended in the presence of ddGTP and ddTTP; the amounts of dideoxynucleotides was adjusted to produce a 3:1 peak height ratio. The pooled fragments were separated on a 4% T LongRanger gel operated at 39 degrees C. Over 500 bases of sequence were generated in 50 min.

Capillary Action↗
PubMed1994

Analysis of polymerase chain reaction-product by capillary electrophoresis with laser-induced fluorescence detection and its application to the diagnosis of medium-chain acyl-coenzyme A dehydrogenase deficiency.

Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) has been developed to detect polymerase chain reaction (PCR) amplified samples. LIF detection was performed using Thiazole Orange as the fluorescent intercalating dye. This method was ca. 100x as sensitive as that with UV detection. The highly sensitive CGE-LIF was applied to the detection of the most prevalent mutation (lysine329-to-glutamic acid substitution) in medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. The disorder, which shows an autosomal recessive inheritance, is known to be highly prevalent among Caucasian population and often mimics as Reye-like syndrome or sudden infant death. A DNA fragment containing the mutation site was PCR-amplified with two sets of allele specific oligonucleotide primers, followed by CGE-LIF. The mutant allele produced a 175-base pairs DNA fragment, which the normal allele generated a 202-base pairs DNA fragment. CGE-LIF clearly distinguished these PCR products, facilitating rapid diagnosis of MCAD deficiency.

Acyl-CoA Dehydrogenase↗
PubMed1994

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis.

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.

Bacteriophage phi X 174↗
PubMed1994

Determination of synthetic food dyes by capillary electrophoresis.

A method for the determination of synthetic tar dyes used as food additives using capillary electrophoresis with photodiode-array detection was investigated. The dyes Erythrosine (R-3), Phloxine (R-104), Rose Bengal (R-105), Acid Red (R-106), Amaranth (R-2), New Coccine (R-102) and Allura Red AC (R-40) were separated on a capillary column (50 cm x 75 microns I.D.) and identified from the absorbance spectra of each peak. The electrophoresis buffer used was a mixture of 25 mM sodium phosphate buffer and 25 mM sodium borate buffer (1:1) (pH 8.0) containing 10 mM sodium dodecyl sulfate (SDS). Substitution of beta-cyclodextrin for SDS in the electrolyte buffer was effective for the separation of R-2 and R-102. This modified method could be employed as an additional assay method for these two dyes.

Amaranth Dye↗