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PubMed1995

Antibody transformation by peroxynitrite as determined using capillary electrophoresis: a feasibility study.

Peroxynitrite may be a physiologically relevant endogenous neurotoxin that forms following CNS trauma when excessive levels of NO and .O2 accumulate. Recently, peroxynitrite was found to inactivate the polyclonal antibody to cAMP. A feasibility study was performed to evaluate the use of capillary electrophoresis as an effective tool regarding the structural transformation of antibody following exposure to peroxynitrite with or without co-incubation with a peroxynitrite scavenger. A polyclonal antibody to cAMP and a monoclonal antibody to plasminogen activation inhibitor-1 were exposed to peroxynitrite with or without penicillamine coincubation. Samples were analyzed by an Applied Biosystems analytical capillary electrophoresis system, model 270A. Initial examination of the peroxynitrite scavenger penicillamine and its reaction with peroxynitrite showed a penicillamine migration peak at about 9.1 min and a presumed s-nitro adduct of penicillamine that migrated at 10.9 min. Exposure of either antibody to peroxynitrite resulted in structural transformation of protein based on changes in migration patterns. In addition, co-incubation with penicillamine prevented this transformation and preserved the pre-peroxynitrite migration patterns of antibodies. In cases of antibody reaction, s-nitro adduct formation could be simultaneously monitored. We found capillary electrophoresis to be ideally suited to this type of analysis. With capillary electrophoresis, we were able to simultaneously monitor the effects of peroxynitrite on large proteins and a small scavenger molecule. As a result, a complete record of the reaction was obtained within a single 15-min analysis period.

Antibodies↗

PubMed1994

Genetic heterogeneity of Helicobacter pylori by pulse-field gel electrophoresis and re-evaluation of DNA homology.

PURPOSE: The genetic heterogeneity of Helicobacter pylori isolates was re-evaluated by using pulse-field gel electrophoresis to examine macrorestriction patterns and by studying DNA homology. MATERIALS AND METHODS: Twenty H. pylori isolates, two closely related species, H. mustelae and H. felis, and Campylobacter spp. were used. Notl-digested macrorestriction patterns were examined by pulse-field gel electrophoresis. DNA homology was examined by the S1 nuclease method, using [3H]-labeled DNA from H. pylori NCTC11637 and two H. pylori isolates for reference. RESULT: Intergenus DNA homology between H. pylori and Campylobacters was 50-60%. Interspecies homology between H. pylori and H. mustelae or H. felis was around 60%. Intraspecies homology among H. pylori isolates was above 80%, except for a few that exhibited 70-80% homology. These findings indicate that all H. pylori isolates were homogeneous and belonged to the same species. Notl pulse-field gel electrophoresis patterns of H. pylori isolates differed markedly at the individual strain level. There was no specific relationship to any deviation from DNA homology, and the differences were observed within rather homogeneous members of the species. CONCLUSION: The polymorphism in the Notl pulse-field gel electrophoresis patterns of H. pylori isolates differed markedly among strains, even though these isolates displayed species homogeneity, with DNA homology of 70-100%.

Campylobacter↗

PubMed1993

Capillary electrophoresis to measure sulfoesterase activity on chondroitin sulfate and heparin derived disaccharides.

Capillary electrophoresis was used to assay sulfoesterase activity on sulfated disaccharides derived from chondroitin sulfate, dermatan sulfate and heparin. The three sulfoesterases studied were chondro-4-O-sulfatase (EC 3.1.6.9) and chondro-6-O-sulfatase (EC 3.1.6.10) from Proteus vulgaris and heparo-2-O-sulfatase from Flavobacterium heparinum. Capillary electrophoresis was used to analyse sulfated disaccharide before and after sulfoesterase treatment and a change in migration time was indicative of the presence of sulfoesterase activity. This assay was used both on purified sulfoesterases and on minor sulfoesterase contaminants present in other enzyme preparations. The high sensitivity of capillary electrophoresis permits the elimination of 35S-radiolabeled substrates normally required to assay sulfoesterases. The high resolution of capillary electrophoresis allows the use of this assay on impure enzyme preparations containing high protein concentrations.

Chondro-4-Sulfatase↗

PubMed1977

[Characterization of Acholeplasma Strains by Horizontal Polyacrylamide Flat Gel Electrophoresis (author's transl)].

Proteins extracted with phenol-acetic acid-water (2:1:0.5, w/v/v) from Acholeplasma laidlawii (PG 8), A. granularum (BTS-39), A. oculi (19L), A. modicum (PG 49), A. axanthum (S743) and the Acholeplasma strains C1 and C112 (which were isolated from aborted horse foetuses) were compared by electrophoresis in horizontal acidic polyacylamide flat gel using the electrophoresis equipment LKB Multiphor 2117. In this system the gels are not prepared in the electrophoresis chamber but between glas plates. For electrophoresis they are applied onto a special cooling plate. This makes it possible to produce a number of identical gels (from the same gel mixture and polymerized under the same conditions) what can be important for comparing investigations. The gels can be stored for more than 4 weeks in the refrigerator at +4 degrees C. Marked differences were observed between the electrophoretic patterns of each of the established species and the horse strains C1 and C112. The results are in agreement with those obtained in serological investigations in which the strains C1 and C112 were different from the established Acholeplasma species.

Acholeplasma↗

PubMed1996

[Separation of methotrexate-polyglutamates by capillary electrophoresis and its application to the measurement of gamma-glutamyl hydrolase activity in human leukemia cells in culture].

We have applied capillary electrophoresis to the separation of methotrexate (MTX)-polyglutamates, and gamma-glutamyl hydrolase (GGH) activities in tumor cells were measured by using this new analytical method. MTX-polyglutamates were sufficiently separated in 15min by capillary electrophoresis with silica fused capillary (phi 50 microns x 75cm), being electrophoresed at 25kV and 30 degrees C in a buffer which contained 20mM sodium tetraborate, 20mM SDS and adjusted pH to 9.5. MTX-polyglutamates eluted were detected at 300nm UV. Cellular extracts obtained from the sensitive and antifolate-resistant human leukemia cell lines, MOLT-3 and K562, were incubated with MTX-glu5 at 37 degrees C for 1, 2 and 4 hr, and the amounts of the degradation products (glu1-glu4) were measured for GGH activity by capillary electrophoresis. There was no significant difference in the production of the metabolites between MOLT-3 and K562 cells (867 +/- 109 vs 799 +/- 56 pmol products/min/1 x 10(7) cells), however, the MTX-resistant MOLT-3 cells with a diminished polyglutamation of folates (MOLT-3/MTX.P-17) and the ZD1694-resistant K562 cells with the impaired membrane transport for reduced folates/MTX/ZD1694 (K562/ZD1694.C) showed decreased activities of GGH (519 +/- 52 and 680 +/- 99 pmol products/min/1 x 10(7) cells, respectively), suggesting the down-regulation of the enzyme in these antifolate-resistant cells concomitant with the intracellular substrate depletion. This study indicates that capillary electrophoresis is a rapid, cost-efficacious method with a sufficient reproducibility in the measurement of GGH activity and must be more suitable for the analysis of clinical samples than HPLC method which requires a large volume of the material.

Down-Regulation↗

PubMed1997

High resolution protein electrophoresis of equine cerebrospinal fluid.

OBJECTIVE: To determine normal CSF electrophoresis patterns in horses, and to determine whether the electrophoretic scans from horses with cervical compression differ from those of neurologically normal horses. ANIMALS: 32 horses assigned to 1 of 2 groups: neurologically normal (n = 18) or cervical compression (n = 14). PROCEDURE: CSF was collected from 18 neurologically normal horses referred to the Marion duPont Scott Equine Medical Center, and protein electrophoresis was performed to describe the normal equine CSF electrophoretogram. Results of CSF electrophoresis from 14 horses with cervical compression were then compared with results for the neurologically normal horses. RESULTS: Horses with cervical compression had decreased beta-globulin fraction, and 1 or 2 prominent post-beta 2 peak(s). When the presence of post-beta peaks was used as a diagnostic criterion for cervical compression, the test had sensitivity of 71.4% and specificity of 81.8%. The positive and negative predictive values were 83.3 and 69.2%, respectively. CONCLUSION AND CLINICAL IMPLICATIONS: Electrophoresis of CSF may be a useful diagnostic aid in evaluation of horses with neurologic disease.

Animals↗

PubMed1998

Detection of oligoclonal bands in patients with neurologic diseases: comparison between agarose gel-, immunofixation- and isoelectric-focusing electrophoresis.

BACKGROUND: The presence of oligoclonal bands (OCBs) in cerebrospinal fluid (CSF) often implies the involvement of the humoral immune response in the disease process. We compared three methods of electrophoresis to determine the frequency and pattern of OCBs in Chinese patients with various neurologic diseases. METHODS: CSF samples and matched serum samples were collected from 122 patients. OCBs were examined in all the CSF samples after agarose gel electrophoresis (AGE) or isoelectric-focusing electrophoresis (IEF); some samples were selected and concentrated for immunofixation electrophoresis (IFE). RESULTS: While 46.7% of the CSF samples showed elevated immunoglobulins using AGE, OCBs were unequivocally identified in 16.4% of samples. In contrast, the detection rate of OCBs using IEF was 54.1%, while that of monoclonal bands was 4.9%. Some OCBs could be detected by IFE, which demonstrated that most of them were IgG-kappa. Using IEF, the sensitivity was 66.7% in multiple sclerosis, 47.8% in myelopathy, 88.9% in chronic inflammatory demyelinating polyradiculoneuropathy, 62.5% in acute inflammatory demyelinating polyradiculoneuropathy, 80.0% in meningoencephalitis and 23.0% in other neurologic diseases. CONCLUSIONS: IEF was the most sensitive method for detecting OCBs. Most patients with type 2 or type 3 patterns of OCBs had multiple sclerosis or meningoencephalitis, although some of these patients may present with a type 4 pattern. Most patients with other diseases had identical patterns of OCBs in both serum and CSF.

Antibody Formation↗

PubMed1999

Nondenaturing electrophoresis. Fractionating of photosynthetic pigment--protein complexes and blood plasma proteins.

Efficient polyacrylamide gel electrophoresis of labile proteins and protein complexes is reviewed. If only 0.001-0.01% SDS is dissolved in the electrode buffers, the detergent does not exhibit denaturing activity and guarantees high quality of electrophoresis. Even the structure and oxygen-producing activity of the labile photosystem PS2 are preserved after electrophoretic separation of photosynthetic pigment--protein complexes from Anacystis nidulans R2 or other cyanobacteria. The overall spectra of absorption or fluorescence of isolated pigment--protein complexes are equal to the corresponding spectra of the photosynthetic membrane. The distribution of chlorophyll molecules between the components of the photosynthetic apparatus coincides in spectral analysis data and gel fraction densitometry. More than 15 electrophoretic fractions of pigment--protein complexes of chloroplasts from green algae and higher plants were observed including some fractions of PS1, some spectrally different forms of light harvesting pigment--protein complexes, and their oligomers. High resolving capacity of electrophoresis was demonstrated by separation of plasma proteins. Low denaturing activity and low thermal dissipation of the electrode buffer solution allow the use of large diameter tubes (3.5 and 8 cm) in polyacrylamide gel electrophoresis. The cell destruction time and the membrane dissolving time are minimized. The method of electrophoretic staining of the gels was tested.

Blood Proteins↗

PubMed1997

Capillary electrophoresis procedures for serum protein analysis: comparison with established techniques.

Methods using automated capillary electrophoresis (CE) instrumentation are available for serum protein electrophoresis with monoclonal band quantitation, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis separations. The advantages of CE over previous gel methods relate to the time and labour saved by the automated instrumentation. High pI monoclonal bands and cryoglobulin specimens can be successfully analysed by CE. However, if the CE application uses a standard company supplied kit, then the cost savings are often negated by the high cost of the kit. Improvements such as the inclusion of both a UV-Vis as well as a fluorescence detector as standard within the one commercial instrument, the production of coated IEF capillaries with a useful life of at least 100 samples, and the introduction of a capillary array into all commercial instrumentation would ensure greater use of CE within both the clinical and other protein laboratories.

Antibodies, Monoclonal↗

PubMed1995

Capillary electrophoresis of human serum proteins and apolipoproteins.

Capillary electrophoresis (CE) analysis of human serum proteins and apolipoproteins is described and compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cellulose acetate membrane electrophoresis. Under optimized CE conditions apolipoproteins I could be determined directly in the serum and quantitated with a linear correlation between peak area and concentration up to a concentration of 100 mg/dL.

Apolipoprotein A-I↗

PubMed1997

Identification of monoclonal proteins in serum: a quantitative comparison of acetate, agarose gel, and capillary electrophoresis.

A selected group of 308 sera were analyzed by capillary electrophoresis (CE), agarose gel electrophoresis (AGE), and cellulose acetate electrophoresis (CAE) and evaluated for abnormalities that would suggest the presence of a monoclonal protein. The sensitivity (an electrophoretic abnormality in sera that contained a monoclonal protein) and specificity (a normal electrophoretic pattern in sera that did not contain a monoclonal protein) was determined for each electrophoretic procedure. CAE was the most specific procedure and CE was the most sensitive. The increase in sensitivity of CE was primarily due to increased detection of cryoglobulins and free light chains. The quantitation of the gamma region and/or monoclonal antibody peaks by CE was similar to results obtained by AGE. Quantitation of very large monoclonal protein peaks (> 3.0 g/dL) by on-line absorption detection (CE) yielded higher results than quantitation by dye-binding (AGE).

Acetic Acid↗

PubMed2003

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis.

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.

Blood Proteins↗

PubMed1987

Preparative displacement electrophoresis (isotachophoresis) of proteins on cellulose columns.

This paper describes the separation of proteins by displacement electrophoresis on columns packed with cellulose powder as a stabilizing medium. Cellulose has virtually no molecular sieving properties and thus differs from dextran, polyacrylamide, and agarose in this respect. Therefore, without the risk of unstacking, columns packed with cellulose permit conventional elution of the protein zones and the use of a counter flow (to increase the effective length of the bed). For the same reason, electroosmotic flow is less disturbing. A continuous elution-migration technique adapted to suit the special requirements of displacement electrophoresis gave better separation than was obtainable by conventional elution. Normal human serum and a fresh hemolysate from human erythrocytes were used as samples. An expression for the volume velocity of the boundaries is derived. This parameter can be used to determine the maximum duration of a run and a suitable pump speed when continuous elution or a counter flow is employed. The special advantages of displacement electrophoresis in cellulose beds are discussed as well as general disadvantages of the displacement technique, including the risk that proteins precipitate during a run.

Blood Protein Electrophoresis↗

PubMed1991

Clinical applications of electrophoresis of human salivary proteins.

Human salivary proteins have been studied by electrophoresis in denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE) as well as by isoelectric focusing (IEF) and two-dimensional procedures, and the clinical applications of this have been reviewed. Whilst non-denaturing PAGE is useful in studying polymorphisms, sodium dodecylsulphate PAGE appears to be otherwise preferable. Immobilized pH gradients containing carrier ampholytes (CAs) give better resolution than CA-based IEF and overcome the problems of cathode drift and loss of basic material. Proline-rich proteins stain poorly with conventional procedures and special techniques are necessary. In clinical studies, findings must be viewed over and above the large number of polymorphisms which occur normally. Studies relating salivary protein and peptide profiles to dental caries susceptibility are encouraging. Specific protein abnormalities have been associated with connective tissue disorders and could form the basis of new non-invasive diagnostic procedures. Protein differences associated with cystic fibrosis and diabetes mellitus, however, merit reinvestigation with the new procedures now available. Detection of HIV antigens in saliva is a new area of research. In the light of new techniques available and new information which has arisen from DNA studies, future prospects for the clinical applications of electrophoresis of saliva look good.

Clinical Laboratory Techniques↗

PubMed2006

Analysis of differential detergent fractions of an AtT-20 cellular homogenate using one- and two-dimensional capillary electrophoresis.

Differential detergent fractionation was used to sequentially extract cytosolic, membrane, nuclear, and cytoskeletal fractions from AtT-20 cells. Extracted components were denatured by sodium dodecyl sulfate (SDS) and then labeled with the fluorogenic reagent 3-(2-furoyl) quinoline-1-carboxaldehyde. Both capillary sieving electrophoresis (CSE) and micellar electrokinetic capillary chromatography (MECC) were used to separate labeled components by one-dimensional (1D) electrophoresis. Labeled components were also separated by two-dimensional (2D) capillary electrophoresis; CSE was employed in the first dimension and MECC in the second dimension. Roughly 150 fractions were transferred from the first to the second capillary for this comprehensive analysis in 2.5 h.

Animals↗

PubMed1983

Genetic variation in proteins: comparison of one-dimensional and two-dimensional gel electrophoresis.

Two proteins with known characteristics on one-dimensional gels were studied by two-dimensional electrophoresis to compare the sensitivities of the two methods in detecting genetic variation. Two-dimensional electrophoresis was found to be less sensitive than several types of one-dimensional gels in distinguishing variants of both proteins. Denaturation of proteins in urea in the two-dimensional method makes it possible to distinguish closely related proteins that differ from each other by units of charge. Many more types of variation in protein sequences can be distinguished on one-dimensional gels in the absence of denaturants. The estimates of heterozygosity based on two-dimensional gels are lower than those based on other methods, at least in part, because of the limited types of sequence differences that can be detected on two-dimensional gels. The application of two-dimensional electrophoresis to the measurement of genetic variation and to the detection of new mutations should be made carefully, in view of the limited sensitivity of the method in finding differences in sequence.

Animals↗

PubMed1989

A systematic study of field inversion gel electrophoresis.

The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first and second dimension. The results are compared with those obtained with other pulsed electrophoresis systems and with a theoretical model.

Bacteriophage lambda↗

PubMed1964

PROTEIN PURIFICATION BY ELUTION CONVECTION ELECTROPHORESIS.

Purified protein components can be recovered from high-resolution gel-electrophoresis patterns by elution-convection electrophoresis. The protein components are all simultaneously eluted from the gel pattern by horizontal electrophoresis and are then simultaneously concentrated by electro-convection to recover the proteins in nearly the original sample concentration. Thirty individual protein fractions can be recovered in 3 hours from a sample containing 200 milligrams of protein.

Convection↗