An improved voltage measurement device for gel electrophoresis in tube apparatus

A device for the measurement of voltage across tube gels was designed and constructed which allows one to measure voltage during electrophoresis without any manipulation of the gel electrophoresis apparatus or gel tube and with the elimination of a source of inaccuracy in previous such devices.     

Use of voltage gradient gel electrophoresis in apoptotic DNA analysis

In this paper the use of voltage gradient gel electrophoresis (VGGE) in the electrophoretic analysis of apoptotic DNAs is described. The peculiarity of VGGE fractionation in enhancing DNA bands in the gel by reducing their thickness was used to obtain a rapid, more selective and higher-quality electrophoretic fractionation of apoptotic DNA with respect to conventional electrophoresis. The use of VGGE fractionations also allowed a reduced amount of DNA to be used to detect a characteristic apoptotic DNA ladder pattern, in a lower agarose gel concentration, with respect to conventional electrophoretic fractionation     

Increasing voltage gradient electrophoresis of DNA

We developed a method which allows electrophoretic fractionation of DNA in an agarose matrix according to an increasing current gradient, using a previously designed [R. Barbieri, V. Izzo, M.A. Costa, G. Giudice, G. Duro, Anal. Biochem. 212 (1993) 168; M.R. Asaro, V. Izzo, R. Barbieri, J. Chromatogr. A 855 (1999) 723] voltage gradient apparatus. This method allows the separation of different DNA fragments by increasing the distances of the components fractionated in the gel, revealing small differences in the length of different DNA components.     

Density gradient electrophoresis of cells in a reversible gel

Density gradient electrophoresis permits the separation of cell types according to surface charge density with high resolution. Any source of flow compromises the resolving power of density gradient electrophoresis. Although procedures have been devised to successfully counteract electroosmotic and convective flows, the final collection of separands requires that they be pumped out of the electrophoresis column. Experiments were therefore designed to test the hypothesis that this flow could also be eliminated by trapping the separated bands in a gel, from which they could be collected by slicing the gel cylinder. Glutaraldehyde-fixed rat and rabbit erythrocytes were used as test particles in a phosphate-buffered isotonic Ficoll-sucrose density gradient in a 2.2 cm diameter, thermostated vertical glass column that could be opened at both ends. Two types of agarose were used as gel polymers: Electrophoresis grade agarose (J.T. Baker Chemical Co.) at final concentrations of 0.1 to 0.25% and SeaPrep ultralow gelling agarose (Marine Colloids Div., FMC Corp.) at a final concentration of 1.0%. Electrophoretic separability of the test particles and fluid stability were tested independently at 55 degrees C and 32 degrees C at which the two agaroses were, respectively, liquid. The experiments demonstrated that the higher temperatures required and the presence of agarose compromised neither the stability of the density gradient nor the migration properties of the cells, and cells can be separated in a sol at a temperature that is compatible with cell viability.     

Two-dimensional gel electrophoresis using immobilized pH gradient tube gels

An apparatus for the preparation of gels for immobilized pH gradient isoelectric focusing (IPG) in glass tubes was developed. Using this apparatus, the highly reproducible immobilized pH gradient can be formed with Immobilines in polyacrylamide gels, and IPG gels at all possible pH ranges can be easily prepared at low cost. The IPG tube gels in the first dimension in two-dimensional gel electrophoresis was used to separate and identify a number of rice embryo proteins in the proteome analysis. There was no difference in resolution of proteins between the tube gels and the commercially available slab gels; after electrophoresis, however, we could efficiently obtain a larger amount of the purified proteins from the tube gels than from the slab gels.     

Polyacrylamide gel electrophoresis of proteoglycans in large-pore gradient gels

A procedure is described for preparing large-pore polyacrylamide gradient gels (1.2–16% T, 10–4% C) that allow the separation of large proteoglycans of molecular mass up to several millions.     

An apparatus for continuous separation of macromolecules by combined electrophoresis and gel filtration

Summary An apparatus for continuous separation of soluble and particulate macromolecules, and of other electrically charged particles by combined electrophoresis and gel filtration, is described. The two principles, electrophoresis and gel filtration, which operate simultaneously at right angles, enable separation between the various macromelecules, depending upon differences in electric charge and molecular wight. The sample to be fractionated is fed continuously into the apparatus while the separated macromolecules reaching the distal end of the instrument are collected at the outlet ports. Data for a continuous fractionation of human plasma proteins using the instrument are given.     

Detection of CFTR mutations using temporal temperature gradient gel electrophoresis

Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is one of the most common autosomal recessive diseases with variable incidences and mutation spectra among different ethnic groups. Current commercially available mutation panels designed for the analysis of known recurrent mutations have a detection rate between 38 to 95%, depending upon the ethnic background of the patient. We describe the application of a novel mutation detection method, temporal temperature gradient gel electrophoresis (TTGE), to the study of the molecular genetics of Hispanic CF patients. TTGE effectively identified numerous rare and novel mutations and polymorphisms. One interesting observation is that the majority of the novel mutations are splice site, frame shift, or nonsense mutations that cause severe clinical phenotypes. Our data demonstrate that screening of the 27 exons and intron/exon junctions of the CFTR gene by TTGE greatly improves the molecular diagnosis of Hispanic CF patients.     

Distinction between supercoiled and linear DNA in transverse agarose pore gradient gel electrophoresis.

Four species of linear DNA and the first four members of a linking series, generated by treatment of plasmid DNA (PUC19, 2.7 kb) with mitochondrial topoisomerase I, were differentiated by transverse agarose pore gradient gel electrophoresis. The experimental curves of migration distance vs. agarose concentration (Ferguson curves) of supercoiled DNA exhibit a steeper trajectory than those of linear DNA of the same size range. As a consequence, the four supercoiled species exhibit an increase in apparent size (relative to linear DNA standards) with increasing agarose concentration. Both the crossing of the Ferguson curves with those of linear standards as well as the apparent size increase with agarose concentration can serve to detect supercoiled plasmid-sized DNA in mixtures with linear DNA.     

Transverse pore gradient gel electrophoresis of lipoprotein [a] using methylenebisacrylamide gradients.

We describe a modification of transverse pore gradient gel electrophoresis in which pore size is regulated by crosslinker proportion (%C) rather than by total monomer concentration (%T). We electrophoresed plasma lipoprotein [a] transversely across linear N,N'-methylenebisacrylamide gradients and measured mobility as a function of %C. This method allows for the simultaneous assessment of pore sizes generated over a wide range of crosslinker proportions.     

Temperature gradient gel electrophoresis: rapid detection of alpha-1-antitrypsin deficiency carriers.

The homozygous state of the alpha-1-antitrypsin (alpha 1AT) deficiency variant Z is associated with severe liver damage in early childhood and progressive lung emphysema in adulthood. A single base transition (G to A in codon 342) in exon V is causing the severe disease. The Glu342 to Lys342 mutation can be detected conventionally by isoelectric focusing (IEF) or on the DNA level by the newly developed method of temperature gradient gel electrophoresis (TGGE). It is the aim of this study to describe the TGGE technique, to compare the results with conventional IEF, and to discuss its efficiency for different diagnostic applications.     

Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.     

Analysis of members of the Ig-gene superfamily by thermal gradient polyacrylamide gel electrophoresis.

A solid aluminum block, connected with a warm and cold thermostated waterbath, provided for a linear transversal temperature gradient (TG) during polyacrylamide gel electrophoresis (PAGE). Noncovalently bound heavy chain dimers as well as heavy-light chain dimers, derived from human monoclonal IgG, could be melted into monomers using a 40-75 degrees C TG under conditions of sodium dodecyl sulfate-PAGE. Using native PAGE, major histocompatibility complex (MHC) class I molecules, preloaded with the iodinated peptide FAPGNYPAL could be melted in a 4-40 degrees C TG to release the peptide. The method is in general applicable to thermal stability analysis of noncovalently bound hetero-oligomers if the product after melting possess different electrophoretic mobilities.     

Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis

Mitochondrial disorders are a group of clinically and genetically heterogeneous diseases. Common recurrent mitochondrial DNA (mtDNA) point mutations account for the molecular defects of a small proportion of patients. In order to identify mtDNA mutations, comprehensive mutational analysis of the entire mitochondrial genome is necessary. We developed the temporal temperature gradient gel electrophoresis (TTGE) method to screen for mutations in mtDNA. The entire mitochondrial genome was amplified using 32 pairs of overlapping primers followed by TTGE analysis of the DNA fragments. TTGE method was first validated on 200 DNA fragments containing known mutations or polymorphisms. On TTGE, homoplasmic nucleotide substitutions show a single band shift and heteroplasmic mutations show multiple banding patterns. The known mutations or polymorphisms were correctly identified. TTGE was then used to screen for unknown mutations in the mitochondrial genome. DNA banding patterns, deviated from wild-type, suggestive of either homoplasmic or heteroplasmic mutations, were followed by direct DNA sequencing to identify the mutations. Numerous mutations and polymorphisms were detected. The results demonstrated that TTGE detects and distinguishes heteroplasmic mutations from homoplasmic polymorphisms. It also detects heteroplasmic changes in the background of a homoplasmic polymorphism. Overall, TTGE was proven to be a simple, rapid, sensitive, and effective mutation detection method.     

Novel cross-linked polyacrylamide matrices: an investigation using gradient gel electrophoresis

Gradient gel electrophoresis was used to examine the separation properties of novel cross-linking compounds for polyacrylamide (PAAm). At low %T and at the same %C protein migration difference is accentuated for bismethacrylamide cross-linked networks relative to bisacrylamide cross-linked networks. Similar properties were observed for cyclic monomers at low %T. This trend is maintained throughout the gradient. However, at higher %T migration differential relative to N,N'-methylenebisacrylamide (Bis) was less pronounced. Evidence from gradient gels suggests that reactivity and functionality of vinyl groups impose an overriding control over network formation.     

Computer analysis of proteins separated by polyacrylamide gradient pore gel electrophoresis

A method for measuring lymph-to-plasma (L/P) protein concentration ratios obtained from protein fractions separated by polyacrylamide gradient gel electrophoresis is presented. A curve-fitting technique is used to decompose lymph and plasma electropherograms containing multiple components into individual components, eliminating protein-protein overlap regions. This allows the concentration of each component in the mixture to be measured accurately, yielding more precise estimates of L/P ratios. This technique consists of three phases. Individual electropherograms are constructed for proteins of various sizes by taking a weighted average of measured electropherograms obtained from the two protein standards closest in size to the protein of interest. Using these generated standard curves, the multicomponent lymph and plasma curves are decomposed into the least number of equally spaced components that yield a good fit. A linear least-squares method is used to do this. Each protein fraction is multiplied by the total measured protein concentration to provide a concentration for each component. Finally, L/P concentration ratios of protein fractions with visible peaks were computed by applying an averaging technique to the equally spaced protein fractions. Plots of sheep lung L/P ratio versus protein size obtained in this manner were compared to L/P ratios obtained using a method of analysis which does not correct for protein overlap. The corrected L/P ratios showed less scatter than the uncorrected curves. Lung lymph data analyzed with the correction method indicated an increased lung microvascular permeability for large proteins following endotoxin infusion, whereas the uncorrected curves were too noisy to support this concept.     

Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin

Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70-80 degrees C in neutral to mild alkaline buffers or at 37 degrees C and slightly acidic pH (6-7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis.