Biomedical subjects

E Gombocz

Publications and source records attributed to E Gombocz.

7 recordsLinked to original sources

PubMed1995

Separation, real-time migration monitoring and selective zone retrieval using a computer controlled system for automated analysis.

High throughput routine analysis of dsDNA fragments or molecular weight determination of proteins via electrophoresis still require significant efforts to obtain results of high reproducibility and accuracy. This paper analyses the use of a fully automated multi-purpose real-time gel electrophoresis system in these applications and evaluates the benefits of this new concept for routine and research. By comparing currently used systems with this new approach, it also addresses the analytical use of information resulting from real-time dynamic migration monitoring via fluorescence photometry over commonly obtained results from post-run fixation, visualization and evaluation at a single time of the separation. The simultaneous separation of components in multi-gel systems, pre-concentration of sample components, and the ability to perform in-gel assays for biological activity are discussed on basis of routine gel separations of restriction enzyme digested DNA fragments, native and denaturing protein separations and enzyme activity determination. Interactive, selective retrieval of separated components in the nano- to microgram range is carried out for real-time isolation of proteins or dsDNA fragments. Results are compared to blotting in respect to ease of use and transfer efficiency and for immediate availability of macromolecules for sequencing or mass spectroscopy. The Windows-based operating software is critically reviewed for functionality, user-friendliness, graphical data representation and GLP compliance for LIMS oriented forensic or certified laboratories. A statistical evaluation of lane-to-lane and gel-to-gel reproducibility of mobility data, quantification and molecular weight determination concludes the paper.

Autoanalysis↗

PubMed1991

Computers.

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Computers↗

PubMed1991

Procedures and computer program for deriving the Ferguson plot from electrophoresis in a single pore gradient gel: application to agarose gel and a polystyrene particle.

This study presents a computerized evaluation of pore gradient gel electrophoretograms to arrive at estimates for both the particle-free mobility and retardation coefficient, which is related to particle size. Agarose pore gradient gels ranging from 0.2 to 1.1% agarose were formed. Gel gradients were stabilized during their formation by a density gradient of 0-20% 5-(N-2,3-dihydroxypropylacetamido)- 2,4,6-triiodo-N,N'bis-(2,3-dihydroxypropyl)-isophthalamide (Nycodenz). Densitometry of gelled-in Bromophenol Blue showed that these pore gradients exhibited a linear central segment and were reproducible. Migration distances of polystyrene sulfate microspheres (36.5 nm radius) in agarose pore gradient gel electrophoresis were determined by time-lapse photography at several durations of electrophoresis. These migration distances were evaluated as a function of migration time as previously reported (D. Tietz, Adv. Electrophoresis 1988, 2, 109-169). Although this is not necessarily required, the mathematical approach used in this study assumed linearity of both the pore gradient and the Ferguson plot for reasons of simplicity. The data evaluation on the basis of the extended Ogston model is incorporated in a user-friendly program, GRADFIT, which is designed for personal computers (Macintosh). The results obtained are compared with (1) conventional electrophoresis using several gels of single concentration with and without Nycodenz, and (ii) a different mathematical approach for the analysis of gradient gels (Rodbard et al., Anal. Biochem. 1971, 40, 135-157). Moreover, a simple procedure for evaluating linear pore gradient gels using linear regression analysis is presented. It is concluded that the values of particle-free mobility and retardation coefficient derived from pore gradient gel electrophoresis using the different mathematical methods are statistically indistinguishable from each other. However, these values are different, albeit close, to those obtained from conventional Ferguson plots. One of the possible reasons for this relatively minor discrepancy is that the particle-free mobility changed slightly during electrophoresis, which has a different effect on electrophoresis in homogeneous gels (single time measurement) and pore gradient gels (multiple time measurements). The characterization of particles according to size and charge by pore gradient electrophoresis provides a significant operational simplification and sample economy compared to that requiring the use of several gel concentrations, although at the price of increased requirements of instrumentation.

Confidence Intervals↗

PubMed1991

Simultaneous Ferguson plot analysis, using electrophoresis on a single agarose pore gradient gel, of DNA fragments contained in a mixture.

A recent study has demonstrated the feasibility of obtaining Ferguson plots in agarose gel electrophoresis, using a single pore gradient gel. We now report three remedies for defects in the previous experimental approach: (i) UV-absorbing media for density stabilization of the gel is avoided by replacing 5-(N-2,3-dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihy droxypropyl) isophthalamide (Nycodenz) with heavy water; this renders the method applicable to ethidium bromide-labeled DNA. (ii) The density stabilizing medium is kept from having an effect on field strength. (iii) Data collection by uninterrupted time-lapse photography is possible by using an apparatus with a quartz window. These three measures make the method practical for the gel electrophoretic identification and physical characterization of DNA species, potentially up to 50 kb in size.

DNA↗

PubMed1989

Program in BASIC for Ferguson plot analysis, using a personal computer: application to gel electrophoresis in a continuous buffer.

A program in BASIC suitable for personal computers is described which is applicable to gel electrophoresis conducted in a single (continuous) buffer. The curve fitting is to a polynomial function, allowing for an objective selection of the most appropriate curve type and order--linear, convex or concave--in the particular application. Results do not differ significantly from previous programs for evaluation of linear Ferguson plots or of curve fitting to an exponential function for evaluating convex plots, executed on mainframe computers such as the DEC-10 (Digital) and IBM 370 computers. Thus, the program combines original versatility with, for the first time, the possibility for widespread application of Ferguson plot analysis on personal computers.

Blood Protein Electrophoresis↗

PubMed1989

Rehydratable agarose gels: use of low EEO agarose without charged additives.

Agarose [SeaKem HGT(P)] gels of 0.5% to 1.2% can be dried at room temperature and rehydrated to 84-85% of their original water content. Mobilities, using the 84-85% rehydrated gels, are indistinguishable from those on the original gels. This suggests that the fiber structure and effective pore size of agarose are independent on the 16% loosely bound (and probably synersed) water.

Cholic Acids↗

PubMed1981

[Immunological estimation of bovine casein in sheep cheese products (author's transl)].

Casein fraction of cow's milk and sheep's milk are very similar in their immunological behavior. Therefore, in the presence of sheep's milk, precipitation reaction with commercially obtained serum are not specific. Results from comparative immunoelectrophoresis according to Grabar and Williams also are equivocal, for heat and other denaturing factors caused shifts in the pattern of just the small group of differing elecrophoretic bonds present among many identical bonds. Qualitative identification of large amounts of cow's milk added to sheep's milk or of bovine casein to sheep cheese or similar products are possible in principle by two dimensional immunoelectrophoresis or so called crossed immunoelectrophoresis as described by Clarke and Freeman, but this estimation takes a lot of time and is not suitable for analysis of a series of samples. Sufficient sensitivity for certain and semiquantitative statements about the content of cows' milk casein in products from sheep's milk are obtained by the technique of radial double diffusion by Ouchterlony in Agarose gels only by the saturation of commercial anti-bovine-casein-serum by means of a suitable protein-standardized sheep's milk extract and its subsequent work-up and lyophilisation to yield a stable serum.

Animals↗