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PubMed2002

Detection of genomic alterations in human endometrial cancer by two-dimensional gel electrophoresis.

OBJECTIVE: The purpose of this study was to detect genomic alterations in human endometrial cancer by two-dimensional gel electrophoresis. STUDY DESIGN: With use of a newly developed two-dimensional gel electrophoresis assay, we scanned 19 high-risk DNA fragments for alterations in human endometrial hyperplasias and adenocarcinomas. This method includes cleaving of high-molecular-weight DNA, radioactive labeling, and separating DNA fragments by two-dimensional gel electrophoresis. By comparing the two-dimensional gel electrophoresis profile (spots) of neoplastic with normal endometrium, genetic alterations such as amplification, allelic loss, and hypermethylation or hypomethylation can be detected. RESULTS: Seven of 8 human endometrial adenocarcinoma (88%) and 1 of 2 hyperplasias (50%) revealed changes in spot density. The number of spots changed per specimen was 4. The median percentage of specimens with changes in an individual spot was 30%. Eleven spots had a reduction or loss of spot density, and 8 spots had an increase in spot density. CONCLUSION: By use of a novel two-dimensional gel electrophoresis assay, we identified genetic alterations in 50% of hyperplasias and 88% of endometrial adenocarcinomas.

Adenocarcinoma↗

PubMed1990

Enhanced resolution of DNA restriction fragments: a procedure by two-dimensional electrophoresis and double-labeling.

A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern. The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension. A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an "x,y" coordinate system with size markers. This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis. To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain. After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 35P. This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis. The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films. Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel. An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map. We have utilized the difference map obtained from Escherichia coli strains HB101 and HB101 (lambda) genomic DNA to show the incorporation of one copy of phage lambda DNA without the use of a lambda DNA probe. This is the same test system that was used previously.

Bacteriophage lambda↗

PubMed1979

Comparison of hemoglobins Wood (alpha 2 beta 2 97 leu) and Malmö (alpha 2 beta 2 97 gln). Diagnostic value of citrate agar electrophoresis.

Diagnostic value of citrate agar electrophoresis. Am J Clin Pathol 71:668-671, 1979. Of approximately three dozen hemoglobin variants that have greater than usual oxygen affinity, nearly half are inseparable from hemoglobin A by electrophoresis at pH 8.6. A comparison of hemoglobins Wood (alpha2beta297leu) and Malmö (alpha2beta297gln) is of interest from several standpoints. They represent similar substitutions at the identical locus in the beta chain. They result in identical clinical and hematologic manifestations. Oxygen affinities of these variants are identical. Both are poorly resolved from hemoglobin A by electrophoresis at pH 8.6. The position of each is identical when studied by isoelectric focusing in polyacrylamide gel. Finally, they are easily distinguished by citrate agar electrophoresis at pH 6.2. The excellent resolution of hemoglobins Malmö and Wood from each other results neither from difference in charge, nor size, nor in quaternary structure. This technic provides a simple but effective means for identifying and differentiating these hemoglobin variants. Comparison with the results of citrate agar electrophoresis of other high oxygen-affinity hemoglobins indicates that the findings for hemoglobins Malmö and Wood are unique and unambiguous.

Adolescent↗

PubMed1979

Study of gammopathies with immunofixation electrophoresis.

Immunofixation electrophoresis and immunoelectrophoresis were compared in 60 samples (51 sera and 9 urines) containing apparently homogenous bands, detected by electrophoresis. To determine the correlation with clinical findings, the patients were divided into three groups: (1) symptomatic with monoclonal immunoelectrophoretic patterns; (2) asymptomatic with monoclonal immunoelectrophoretic patterns; (3) asymptomatic with polyclonal immunoelectrophoretic patterns. The results from immunoelectrophoresis and immunofixation electrophoresis were consistent with each other in all cases of Groups I and III in terms of clonality (i.e., whether monoclonal or polyclonal) and immunoglobulin class; whereas in Group II, which was composed of asymptomatic patients, two sera and three urines were identified to have monoclonal changes by immunoelectrophoresis but polyclonal changes by immunofixation electrophoresis. It is recommended that immunoelectrophoresis still be used for routine clinical service, but supplemented by immunofixation electrophoresis in equivocal cases, namely: (1) light-chain changes masked by an umbrella effect of immunoglobulin G (IgG); (2) abnormal bands located in atypical areas or overlapped with a normal serum protein; (3) a normal-looking "free" light chain present in the urine, mimicking Bence Jones protein; (4) controversial cases of biclonal gammopathy; (5) mini-monoclonal or oligoclonal protein bands; (6) immune complexes.

Bence Jones Protein↗

PubMed1992

Electrophoresis underestimates the concentration of polyclonal immunoglobulins in serum.

The concentration of gamma-migrating immunoglobulins in blood serum was measured in 200 healthy blood donors by two methods, nephelometry and electrophoresis. Nephelometric values were calculated as IgG + IgM + 1/2 IgA because immunoglobulin G (IgG) and immunoglobulin M (IgM) migrate entirely within the gamma region and immunoglobulin A (IgA) is split between the gamma and beta regions. Electrophoretic values were calculated as % gamma x total protein. Gels were scanned with two different densitometers, providing two sets of electrophoretic data. In each case, the correlation between nephelometry and electrophoresis was very good (r = 0.94 and r = 0.96). However, electrophoresis consistently gave lower results than nephelometry (mean = 2.3 g/L and 4.8 g/L lower, respectively). The disparity between these methods was greater as the concentration of immunoglobulins increased. It is concluded that (1) electrophoresis gives a relative but not absolute value of immunoglobulin concentration in serum; (2) nephelometric and electrophoretic values cannot be used interchangeably; and (3) reference ranges for electrophoresis must take into account the densitometer used.

Adolescent↗

PubMed1988

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.

Animals↗

PubMed1996

Capillary electrophoresis for drug analysis in body fluids.

Capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) represent attractive methods for the determination of drugs and metabolites in body fluids. In CZE, minute (nanoliter) quantities of samples are applied to the beginning of a fused-silica capillary filled with buffer. On application of a high-voltage DC field, charged solutes begin to separate and are swept through the capillary by the combined action of electrophoresis and electroosmotic bulk flow and are on-column detected toward the capillary end. In MECC, the buffer contains charged micelles (e.g., dodecyl sulfate micelles) and both uncharged and charged solutes separate based on differential partitioning between the micelles and the surrounding buffer and, if charged, also by differential charge effects, including electrophoresis. Based on validated MECC drug assays developed in our laboratory, key aspects of measuring drug levels by MECC, including sample preparation, solute detection and identification, quantitation, reproducibility, and quality assurance are discussed. Drug levels determined by MECC are shown to be in good agreement with those obtained by nonisotopic immunoassays and/or high-performance liquid chromatography (HPLC). Using on-column multi-wavelength detection, this technology is also well suited for toxicological drug screening and confirmation and for the exploration of drug metabolism. Compared with HPLC and gas chromatography, capillary electrophoresis has distinct advantages, including automation, small sample size, minimal sample preparation, use of very small amounts of organic solvents and inexpensive chemicals, ease of buffer change and method development, and low cost of capillary columns. Electrokinetic capillary assays are complementary to the widely employed immunoassays. The state of the art and the pros and cons of capillary electrophoresis for the determination of drugs in body fluids are discussed with the goal of encouraging newcomers to start using this emerging analytical methodology.

Body Fluids↗

PubMed1981

Further studies on bovine serum amylase. 2. Affinity electrophoresis.

The polymorphism of bovine serum amylase, which is controlled by the AmI locus, has previously only been demonstrated by starch gel electrophoresis. The addition of maltose to starch gels has been demonstrated to inhibit any subsequent separation of the AmI isozymes by starch gel electrophoresis. When electrophoresis was conducted in a support medium in the absence of starch no polymorphic variation was detected amongst samples from animals of different AmI phenotypes. The addition of starch to agarose gels has been shown to facilitate the subsequent detection of the AmI polymorphism by agarose/starch gel electrophoresis. The electrophoretic resolution of the AmI isozymes has been demonstrated to depend upon differences in affinity for starch rather than differences in net charge. The starch gel electrophoretic separation of the AmI isozymes is, therefore, another example of affinity electrophoresis. All the AmI amylases have been shown to share a common isoelectric point of pH 3.5.

Amylases↗

PubMed1983

Aberrant migration of lipopolysaccharide in sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

Purified lipopolysaccharides of salmonellae strains were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Pre-electrophoresis of polyacrylamide gels had no apparent effect on one-dimensional silver-stained lipopolysaccharide profiles. However, without pre-electrophoresis, two-dimensional and three-dimensional patterns contained numerous bands with varied migration patterns compared to those in the one-dimension gels. The lipopolysaccharide was altered within the polyacrylamide gel during electrophoresis. Pre-electrophoresis of gels eliminated aberrant migration patterns.

Electrophoresis, Polyacrylamide Gel↗

PubMed1972

Characterization and comparison of mycobacterial antigens by two-dimensional polyacrylamide gel electrophoresis.

Cell extracts and culture filtrates were prepared from several mycobacterial species and subjected to two-dimensional acrylamide gel electrophoresis. The cell preparations were first electrophoresed in a 7% homogeneous gel followed by a second electrophoresis, at right angles to the first separation, in a 2 to 30% linear gel gradient slab. Because the stained slab resembled a "fingerprint" or "peptide map," this procedure has been called "electroprotein mapping." More than 200 protein-staining spots were detected in several of the cell extracts, each map being characteristic of the species examined. The single most characteristic portion of each map was the region between the 16 and 30% gel concentrations. This region consisted of several small-molecular-weight components (many of peptide proportions) which together appeared as the most anodic band in the 7% gel column (first separation). The number and location of these components in the linear gel slab (second separation) were specific for each species tested. By electrophoresis of molecular-weight markers (i.e., albumin, myoglobin, cytochrome c) with and across (transverse electrophoresis) the gel gradient, the relative molecular weights of the unknown cell extract components could be estimated. These results suggest that two-dimensional acrylamide gel electrophoresis is a useful tool for the characterization of mycobacterial cell constituents.

Antigens, Bacterial↗

PubMed1995

Comparison of MIDI Sherlock system and pulsed-field gel electrophoresis in characterizing strains of methicillin-resistant Staphylococcus aureus from a recent hospital outbreak.

An outbreak of methicillin-resistant Staphylococcus aureus infections at the University of Utah Health Sciences Center occurred over a 7-month period. While the isolates phenotypically appeared to be similar in gross morphology and have similar Vitek antibiotic susceptibility patterns, two additional methods of strain characterization were evaluated to enhance the epidemiological investigation: pulsed-field gel electrophoresis and gas chromatography with the MIDI Sherlock system. Sherlock uses gas chromatography to qualitatively and quantitatively analyze the cellular fatty acid composition of organisms and creates two-dimensional plots based on principal-component analysis to define groups of closely related organisms. All isolates were also evaluated by digesting their chromosomal DNAs with the low-frequency-cutting enzyme SmaI and separating the restriction fragments by contour-clamped homogeneous electric field gel electrophoresis. Sample preparation for this pulsed-field gel electrophoresis included a novel cell lysis procedure involving achromopeptidase, greatly reducing the turnaround time. Isolates tested were recovered from the following: 45 suspected outbreak patients, 6 hospitalized patients believed to be unrelated to the outbreak, 6 patients from outside the hospital, and one health care practitioner implicated in the outbreak. Of 45 phenotypically similar suspect strains, 43 clustered tightly on the Sherlock two-dimensional plot. All outbreak patient isolates were also identical by pulsed-field gel electrophoresis with the exception of the same two outliers identified by Sherlock. In this epidemiologic investigation, we found an excellent correlation between the Sherlock and pulsed-field gel electrophoresis results for strain characterization of methicillin-resistant S. aureus.

Bacterial Typing Techniques↗

PubMed1997

Lectin affinity electrophoresis in a yolk sac tumour in the vagina with yolk sac tumour-type glycoform of alpha fetoprotein.

AIMS: To investigate a potential diagnostic use of alpha fetoprotein (alpha FP) isoform analysis by lectin affinity electrophoresis to distinguish between endodermal sinus tumours arising in the vagina in infants from those at other sites. METHODS: alpha FP in the serum of a patient with a vaginal endodermal sinus tumour was analysed for its isoforms by lectin affinity electrophoresis. The isoforms were compared with that of cord serum, sera of hepatoid adenocarcinoma of the uterus, and endodermal sinus tumour of the ovary. RESULTS: The isoforms of alpha FP obtained by lectin affinity electrophoresis in the serum of the patient with vaginal endodermal sinus tumour differed from the isoforms of alpha FP in the cord serum of normal neonates, and sera of patients with hepatoid adenocarcinoma of the uterus or endodermal sinus tumour of the ovary. CONCLUSIONS: Endodermal sinus tumour arising in the vagina could be distinguished from that in the ovary by the lectin affinity electrophoresis, and a potential diagnostic use of alpha FP isoform analysis by the lectin affinity electrophoresis for the detection of the endodermal sinus tumour in infants was demonstrated.

Diagnosis, Differential↗

PubMed2004

An XML standard for the dissemination of annotated 2D gel electrophoresis data complemented with mass spectrometry results.

BACKGROUND: Many proteomics initiatives require a seamless bioinformatics integration of a range of analytical steps between sample collection and systems modeling immediately assessable to the participants involved in the process. Proteomics profiling by 2D gel electrophoresis to the putative identification of differentially expressed proteins by comparison of mass spectrometry results with reference databases, includes many components of sample processing, not just analysis and interpretation, are regularly revisited and updated. In order for such updates and dissemination of data, a suitable data structure is needed. However, there are no such data structures currently available for the storing of data for multiple gels generated through a single proteomic experiments in a single XML file. This paper proposes a data structure based on XML standards to fill the void that exists between data generated by proteomics experiments and storing of data. RESULTS: In order to address the resulting procedural fluidity we have adopted and implemented a data model centered on the concept of annotated gel (AG) as the format for delivery and management of 2D Gel electrophoresis results. An eXtensible Markup Language (XML) schema is proposed to manage, analyze and disseminate annotated 2D Gel electrophoresis results. The structure of AG objects is formally represented using XML, resulting in the definition of the AGML syntax presented here. CONCLUSION: The proposed schema accommodates data on the electrophoresis results as well as the mass-spectrometry analysis of selected gel spots. A web-based software library is being developed to handle data storage, analysis and graphic representation. Computational tools described will be made available at http://bioinformatics.musc.edu/agml. Our development of AGML provides a simple data structure for storing 2D gel electrophoresis data.

Computational Biology↗

PubMed2006

Beverage-agarose gel electrophoresis: an inquiry-based laboratory exercise with virtual adaptation.

A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As such, we chose it as a platform to expose high school and undergraduate students to the active process of scientific inquiry in general, while specifically teaching electrophoresis. First, we optimized DNA electrophoresis in the laboratory by using common beverages instead of standard media (e.g., Tris-based media). Second, we adapted this laboratory process of progressive optimization to a Web-based format in which students had to achieve all the same steps of optimization by performing serial electrophoreses. And third, we evaluated the use of this entirely Web-based virtual laboratory exercise in high school and undergraduate biology courses. Students learned fundamental and practical principles of electrophoresis, while experiencing the essential inquiry-based process of optimizing a technique, and they also enjoyed it. Our findings provide a readily accessible, inexpensive, and intriguing technique for teaching electrophoresis and the progressive optimization of a laboratory technique.

Beverages↗

PubMed2003

The use of capillary electrophoresis for DNA polymorphism analysis.

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.

DNA Mutational Analysis↗

PubMed1993

An immunoblotting procedure following agarose gel electrophoresis for subclass typing of IgG paraproteins in human sera.

A method for subclass typing of IgG paraproteins in human sera following agarose gel electrophoresis is presented. After electrophoretic separation, serum proteins were blotted by capillary diffusion onto nitrocellulose. Mouse anti-human IgG1-IgG4 monoclonal antibodies were exposed to bound IgG. Goat anti-mouse IgG alkaline phosphatase conjugate was employed as second antibody. Thirty six sera were examined, in which the presence of an IgG paraprotein had previously been proved by agarose gel electrophoresis and subsequent immunofixation with anti-IgG antiserum. The subclass frequency distribution was 27 IgG1, 6 IgG2, 2 IgG3 and 1 IgG4. By means of IgG subclass typing in 8 out of these 36 sera, a total of 12 additional monoclonal bands (1 to 2 bands per serum) were detected which were not seen after immunofixation electrophoresis with anti-IgG antiserum. Eleven of 12 additional bands belonged to an IgG subclass different from that of the bands already detected by immunofixation electrophoresis. Light chain typing was performed in 9 of 12 bands found additionally. Two of these 9 bands belonged to a light chain class different from that of the bands already detected by immunofixation electrophoresis. The method described can be employed to further elucidate the possible diagnostic and prognostic significance of the subclass type of an IgG paraprotein.

Adult↗

PubMed2005

Recent advances in 2D electrophoresis: an array of possibilities.

2D electrophoresis is currently the most widespread technique used for performing functional proteomics (i.e., the large-scale analysis of alterations in protein expression levels). Nevertheless, several limitations inherent to this technology have restricted the full potential of this protein differential display methodology for years. This has even led to the abandonment of 2D electrophoresis by several groups that switched to performing gel-free functional proteomics analyses based on liquid chromatography and mass spectrometry. Meanwhile, important recent advances in 2D electrophoresis, such as the introduction of fluorescent 2D difference gel electrophoresis and numerous protein prefractionation techniques, have thoroughly modernized 2D electrophoresis, making it again one of the preferred methods for the analysis of protein expression differences in many laboratories.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2000

Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat islets of Langerhans.

Interleukin-1beta (IL-1beta) treatment of neonatal rat islets for 24 h induces changes in the expression of 105 of 2,200 proteins, as determined previously by two-dimensional (2D) gel electrophoresis. Nitric oxide (NO) has been implicated as one of the mediators of IL-1beta effects in insulin-containing cell lines and rat islets. The aims of this study were 1) to determine the involvement of NO in IL-1beta-induced alterations in protein expression and 2) to investigate the effects of chemically generated NO on protein expression by 2D gel electrophoresis of neonatal rat islet samples. IL-1beta-induced NO production was prevented by incubation of islets in arginine-free medium supplemented with the arginine analog NG-nitro-L-arginine. [35S]methionine-labeled islet proteins were separated using 2D gel electrophoresis and analyzed using the BioImage computer program. Analysis revealed that the expression levels of 23 protein spots of the 105 protein spots, altered by prior treatment with IL-1beta (60 U/ml) alone, were significantly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production was prevented. The effects of chemically generated NO were investigated by exposing islets to the NO donor GSNO (100 micromol/l) for 24 h before labeling with [35S]methionine and 2D gel electrophoresis. Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectable proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expression of up to 42 proteins is altered by cytokine-induced or chemically generated NO in the precise experimental conditions chosen and 2) that the majority of proteins altered by prior treatment with IL-1beta may be the result of NO-independent IL-1beta-mediated regulation of gene expression. This study demonstrates that the combination of 2D gel electrophoresis and mass spectrometry is a powerful tool in the identification of beta-cell proteins involved in the response to toxic mediators.

Animals↗