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PubMed1994

Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis.

Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis. The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used. The purpose of the present work was to characterize and compare this new isolate with reference strains of R. quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis. SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R. quintana. However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera. Pulsed-field gel electrophoresis allowed differentiation of the French R. quintana isolate from R. quintana Fuller and may serve as an epidemiological tool.

AIDS-Related Opportunistic Infections↗

PubMed1999

Development of capillary electrophoresis as an alternative to high resolution agarose electrophoresis for the diagnosis of multiple sclerosis.

The presence of oligoclonal bands in cerebrospinal fluid (CSF) is used as a diagnostic indicator of multiple sclerosis (MS). These bands, gamma-globulins thought to result from a restricted antibody response directed against autoantigens or viral antigens, are consistent with CSF-specific immunoglobulin synthesis when observed in the spinal fluid and not in the serum. Current methodology commonly involves electrophoresing concentrated CSF with high-resolution agarose gel electrophoresis (HRAGE) followed by protein staining in order to visualize the oligoclonal bands. Capillary zone electrophoresis (CZE) was evaluated as an alternative method. Separation of CSF and serum proteins from 54 patients in a bare silica capillary containing a high pH borate buffer allowed for resolution of the five major zones including the gamma-region and showed a 90% concordance with the results from HRAGE banding studies. Since a simple borate buffer did not provide adequate resolution of the oligoclonal bands in the gamma-region, the separation buffer was augmented with polyethylene glycol (PEG) which provided a significant enhancement in resolution of proteins in this region (24 patient study). In addition to obtaining banding information from electropherograms obtained with these separation conditions, it was feasible to calculate a CSF Index which compared favorably with the results from nephelometry. Finally, we show that zwitterionic additives such as O-phosphorylethanolamine may further enhance resolution and that capallary electrophoresis (CE) may allow oligoclonal banding information to be obtained directly from CSF without concentration.

Buffers↗

PubMed1990

Application of a new electrophoresis technique (2D cryostat section electrophoresis) to synovial tissue of RA patients and comparison with immunohistochemical staining methods.

We describe a new two dimensional electrophoresis technique which is based on the combination of cryostat section technology and IEF- and SDS-gel electrophoresis. The optimal conditions for two dimensional cryostat section electrophoresis are investigated. The application of this technique to synovial membranes of patients suffering from rheumatoid arthritis and osteoarthritis is described.

Arthritis, Rheumatoid↗

PubMed1984

Two-dimensional electrophoresis of proteins in human serum: improved resolution by use of narrow pH gradients and prolonged electrophoresis.

The limited resolution of serum proteins achieved with a simplified technique of two-dimensional electrophoresis (Clin. Chim. Acta 103: 51-59, 1980) has been improved by using different ampholyte (Ampholine) mixtures in the first dimension, to obtain relatively shallow pH gradients, and prolonged electrophoresis time in the second dimension. The technique has been further simplified, without negative effect, by decreasing the concentration of non-ionic detergent (first dimension), omitting both sodium dodecyl sulfate equilibration and the use of a stacking gel (second dimension), and by using an improved silver-staining procedure. Reverse-polarity isoelectric focusing and non-equilibrium pH-gradient electrophoresis have been successfully used to resolve the basic polypeptides in serum after deleting sodium dodecyl sulfate from the sample preparation. These combined techniques reveal over 1100 polypeptides in human serum. After immunodeletion of albumin, additional serum polypeptides are seen. Immunodeletion of serum proteins from plasma reveals polypeptides that are relatively specific to plasma.

Ampholyte Mixtures↗

PubMed1998

Multicenter evaluation of the Paragon CZE 2000 capillary zone electrophoresis system for serum protein electrophoresis and monoclonal component typing.

Serum protein electrophoresis and typing of monoclonal components (MCs) are routine but time-consuming and technically demanding assays. We evaluated capillary electrophoresis (Paragon CZE 2000) for automation of the two assays. CZE and cellulose acetate electrophoresis gave similar data on 794 samples. Within-run and between-run CVs were < 2% for albumin and gamma-globulins and 4-7% for alpha 1-, alpha 2-, and beta-globulins. Bilirubin, hemoglobin, triglycerides, and fibrinogen were found not to interfere. No carryover by capillaries was detected. The detection limit for MC was < 0.5 g/L. MC assessment by immunosubtraction on 403 samples identified the monoclonal type in all samples with peak concentrations > 10 g/L; only 50% of MCs that could not be quantified by densitometric scan were typed.

Antibodies, Monoclonal↗

PubMed2000

Fast separation of oligonucleotide and triplet repeat DNA on a microfabricated capillary electrophoresis device and capillary electrophoresis.

A laser-induced fluorescence detection system coupled with a highly sensitive silicon-intensified target (SIT) camera is successfully applied to the imaging of a band for DNA fragment labeling by fluorescence dye in a microchannel, and to the visualizing of the separation process on a microfabricated chip. We demonstrated that an only 6 mm separation channel is sufficient for the separation of triplet repeat DNA fragment and DNA molecular marker within only 12 s. The separation using the microfabricated capillary electrophoresis device is confirmed to be at least 18 times faster than the same separation carried out by conventional capillary electrophoresis with 24.5 cm effective length. The use of a short capillary with 8.5 cm effective length is also efficient for fast separation of DNA; however, the microchip technology is even faster than capillary electrophoresis using a short capillary.

DNA↗

PubMed2001

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy.

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).

Chromatography, High Pressure Liquid↗

PubMed1989

Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and western blotting of urinary proteins.

The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories.

Blood Proteins↗

PubMed1990

Free flow electrophoresis for the purification of proteins: I. Zone electrophoresis and isotachophoresis.

The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97% alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.

Alcohol Dehydrogenase↗

PubMed1990

Correlation of capillary zone electrophoresis with continuous free-flow zone electrophoresis: application to the analysis and purification of synthetic growth hormone releasing peptide.

Two carrier-free electrophoretic separation methods, capillary zone electrophoresis (CZE) and continuous free-flow zone electrophoresis (FFZE), have been applied to both microanalysis at the nanogram level and preparative fractionation, with a throughput of 30 mg/h, of synthetic growth hormone releasing peptide (GHRP). A crude product of GHRP, a hexapeptide with the sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, synthesized by the solid phase methodology, was desalted and analyzed by CZE. Based on the results of analytical CZE the separation was converted into a preparative purification procedure by continuous FFZE, employing the same separation medium (0.5 mol/L acetic acid, pH 2.6). The purifity of peptide fractions obtained by FFZE was reevaluated by CZE. The combination of these two techniques proved to be a valuable tool for both peptide analysis and peptide purification. A close correlation of CZE and FFZE, resulting from the fact that both methods are based on the same separation principle (zone electrophoresis) and that both are performed in a free solution of the same composition, was confirmed. However, when transforming data from CZE to FFZE, the different electroosmotic flow, temperature and electric field intensity in the capillary and in the flow-through cell, respectively, have to be taken into account and corresponding corrections have to be made.

Amino Acid Sequence↗

PubMed1994

Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.

Blue native Electrophoresis is a "charge shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. In combination with a second dimension sodium dodecylsulfate electrophoresis it provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, of subunit composition, and of degree of purity and for the detection of subcomplexes. The method was applied to analysis of cytochrome bc/bf complexes. By combination of a novel colorless native polyacrylamide gel electrophoresis (CN-PAGE) with blue native BN-PAGE, a two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes.

Animals↗

PubMed1994

Comparison of the separation of proteins by sodium dodecyl sulfate-slab gel electrophoresis and capillary sodium dodecyl sulfate-gel electrophoresis.

Capillary sodium dodecyl sulfate-gel electrophoresis, a one-dimensional version of the well-established planar analytical method of SDS-polyacrylamide gel electrophoresis, has proven a powerful new microanalytical method for the separation of protein molecules according to their size. In this paper the planar SDS-PAGE and the capillary SDS-gel electrophoresis of different proteins are compared with respect to performance, precision, and efficiency. Molecular weight versus migration distance and migration time curves for 65 proteins, running from 14,200 to 205,000 Da, are given for both methods.

Capillary Action↗

PubMed1996

Immobilized metal ion affinity capillary electrophoresis of proteins--a model for affinity capillary electrophoresis using soluble polymer-supported ligands.

An affinity capillary electrophoresis method has been developed that employs small ligands covalently bound to a replaceable soluble polymer matrix. The metal chelate iminodiacetate-Cu(II) coupled to polyethylene glycol was used as a model system and the interactions of different model proteins, namely ribonucleases, cytochromes c, chymotrypsin, and kallikrein, were investigated. The method allowed for easy determination of dissociation constants using a modified Langmuir adsorption isotherm equation which is applicable to interactions with fast on/off kinetics. It is shown that the general rules for protein interaction with matrix-bound metal chelates established for immobilized metal ion affinity chromatography (IMAC) are maintained in immobilized metal ion affinity capillary electrophoresis (IMACE). In contrast to gel electrophoresis, IMACE allowed for using similar conditions as in IMAC, especially regarding the high salt concentrations, usually employed with the latter technique. The usefulness of the method for quantification of low-affinity interactions and studying protein surface structure and structure/function relationship using the metal affinity is demonstrated.

Animals↗

PubMed2004

Electrophoresis time impacts the denaturing gradient gel electrophoresis-based assessment of bacterial community structure.

We investigated the impact of denaturing gradient gel electrophoresis (DGGE) run time on the assessment of bacterial community structure. Results indicated that increased electrophoresis run time (while maintaining 1000 volt-hours) resulted in dissimilar profiles, likely due to instability of the denaturing gradient. We recommend that DGGE run times be minimized to provide optimal band resolution, as extended electrophoresis times can greatly impact subsequent band-based analyses.

Electrophoresis, Polyacrylamide Gel↗

PubMed1999

Separation of bovine serum albumin and its monoclonal antibody from their immunocomplexes by sodium dodecyl sulfate-capillary gel electrophoresis and its application in capillary electrophoresis-based immunoassay.

A non-competitive immunoassay was performed by sodium dodecyl sulfate-capillary gel electrophoresis with UV detection using bovine serum albumin (BSA) and monoclonal anti-BSA. BSA, anti-BSA and their immunocomplexes were well resolved under non-denaturing conditions. A linear calibration curve was obtained and can be used for the quantification of anti-BSA. The limit of detection of anti-BSA was 0.1 microM under the present conditions. Compared with capillary zone electrophoresis, we believed that this method has the potential to be used as a more general format for performing capillary electrophoresis-based immunoassay of medium- and large-sized analytes.

Antibodies, Monoclonal↗

PubMed2000

Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions.

A set of Escherichia coli freshwater isolates was chosen to compare the effectiveness of denaturing gradient gel electrophoresis (DGGE) vs temporal temperature gradient gel electrophoresis (TTGE) for separating homologous amplicons from the respective uidA region differing in one to seven single base substitutions. Both methods revealed congruent results but DGGE showed a five to eight times higher spatial separation of the uidA amplicons as compared with TTGE, although the experiments were performed at comparable denaturing gradients. In contrast to TTGE, DGGE displayed clear and focused bands. The results strongly indicated a significantly higher discrimination efficiency of the spatial chemical denaturing gradient as compared with the temporal temperature denaturing gradient for separating the uidA amplicons. Denaturing gradient gel electrophoresis proved to be highly efficient in the differentiation of E. coli uidA sequence types.

Base Sequence↗

PubMed1976

Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. II. Plasma membrane proteins analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional immune electrophoresis.

The plasma membrane proteins of lymphocyte populations from normal outbred Syrian hamsters were compared with those of a neoplastic transformant line (GD 248) induced by simian virus 40. Both quantitative and qualitative differences were observed. Gradient dodecyl sulfate-polyacrylamide gel electrophoresis revealed 12 major protein components in the membranes of both cell populations. Both membrane categories also contained small amounts of immunoglobulin. Compared with the membranes of the reference cell population, GD 248 membranes showed a 60% decrease of approximately 210,000 daltons of glycoprotein; a 10% reduction of about a 48,000-dalton band and virtually complete loss of a 15,000-dalton component concomitant with a 57% increase in a 52,000-dalton band; fusion to two subcomponents (mol wt approximately 250,000 daltons); and emergence of approximately 120,000 and 30,000 daltons glycoproteins. In addition, the relative mobility of an approximately 95,000-dalton component increased by roughly 0.02 U. Crossed immune electrophoresis in Trition X-100 with heterologous antiserum against GD 248 microsomal membranes revealed both a new component with a high level of electrophoretic mobility and intensification and additional heterogeneity in a strongly antigenic component with a low level of electrophoretic mobility. Crossed-line immune electrophoresis indicated that at least two antigens in the membranes of GD 248 cells lacked the membranes of the reference cell population.

Animals↗

PubMedDate not supplied

Migration properties of circular DNAs using orthogonal-field-alternation gel electrophoresis.

The migration of a series of supercoiled plasmids ranging in size from 4 to 91 kilobases (kb) has been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter OFAGE gels and are resolved over the same region of the gel as linear DNAs from 260 to 2200 kb. Furthermore, a distinct triphasic migration pattern was observed for the supercoiled DNAs. The migration of plasmids between 6 and 20, and 60 and 91 kb is inversely proportional to size, whereas the mobilities of plasmids between 20 and 60 kb increase with size. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a broad range of pulse times, from 10 to 120s. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of small plasmids shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.

Animals↗