The HPGE 1000 System from LabIntelligence is a fully automated, computer-controlled real-time gel electrophoresis system for the life sciences. LabIntelligence is located in Belmont, California, U.S.A. For more information, call (+1) 650-654-9908 ! 
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[1/1]
Gombocz, E., Cortez, E. and Cole, S.: Rapid Molecular Weight Determination and Quantification of Proteins Using an Automated Electrophoresis System. Biomed. Prod. 18 (8): 36 (1993).

Protein molecular weight determination by manual SDS-PAGE is a widely used bioanalytical technique that has several drawbacks: it is labor-intensive, time consuming, and relatively imprecise. The automated HPGE 1000A High Performance Gel Electrophoresis System significantly improves the technique by making it easier, faster and more accurate. The HPGE 1000A is a completely integrated system. It maintains thermal, electrical and buffer conditions during separation and detects zone migration by fluorescence photometry dynamically in real-time. Easy-to-use software allows for rapid protein molecular weight determination and peak quantitation. A denaturing protocol is presented, which uses a continuous SDS Tris/Tricine buffer system rather than commonly used discontinuous systems. Furthermore, separation is carried out on a 5% T MetaPhor high resolution agarose gel instead of polyacrylamide without compromising results. Scan and peak data illustrate the quality of the separation and the output of the system's peak search function. The variants Phosphorylase A and B, which are typically not separated using manual electrophoresis apparatus, are clearly separated. The conditions described allow for sample loads of approximately 25 ng to 2.5 mg per zone. Mobility data of the reference lane was used to create a calibration curve. The fit resulted in a first-order function, indicating linearity, for log(MW) vs. absolute mobility within the size range indicated. Compared with the manufacturer's declared values, the calculated molecular weights had a relative error of less than 1.0%. These calculations were based on absolute mobilities rather than on relative mobilities (Rf). Migration distance data taken at different points during the run illustrate the reproducibility of the separation from lane to lane independent of the progress of the separation. Using the described protocol, the HPGE 1000A performed electrophoretic separation and complete evaluation of proteins in the range of 14-95 kD in less than 45 minutes with no post-run treatment of the gel. The benefits of the system include fast, accurate, quantitative results over the most common protein size range, the ability to load a broad range of sample concentrations, and the use of easy- to-cast, non-toxic gels.

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[1/3]
Gombocz, E. and Cortez, E.: Separation, Real-Time Migration Monitoring and Selective Zone Retrieval Using a Computer Controlled System for Automated Analysis. Appl. Theor. Electrophoresis 4 (4): 197-209 (1995).

High throughput routine analysis of dsDNA fragments or molecular weight determination of proteins via gel electrophoresis still require significant efforts to maintain 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 concepts 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 for in-gel assaying are discussed on basis of routine gels for restriction enzyme DNA fragments and human serum protein enzyme activity determination. Interactive, selective retrieval of separated components in the nano- to microgram range is carried out for dsDNA fragment isolation and compared to blotting regarding ease of use and transfer efficiency. The Windows-based operating software is critically reviewed for functionality, user-friendlyness, graphical presentation 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.

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[1/4]
Gombocz, E., Cortez, E., Yang, L. and Rammler, D.: HPGE: Fast, efficient retrieval of pure proteins for MALDI-TOF mass spectrometry. Biomed.Prod. 21 (1): 134-135 (1996).).

Transfer and recovery techniques like gel blotting or electroelution of gel slices after the run lack appeal, because they involve labor-intense processes, low and unequal transfer efficiencies and loss of biological activity of the target proteins. A unique alternative to this procedure was introduced recently, which performs zone-selective, vertical electroelution of biologically intact, unlabeled proteins from a horizontal electrophoretic separation directly into small containers for mass spectrometric analysis. This method uses a fully automated gel electrophoresis system with real-time fluorescence photometric determination of the transfer efficiency, which allows immediate use of the eluate for confirming mass spectrometry without additional treatment. The HPGE 1000 High Performance Gel Electrophoresis system consists of a computer-controlled instrument to perform electrophoretic protocols under parameter feedback control, which dynamically monitors zone migration. Therefore, the user is able to identify and extract any separated component at any time during the run in a single-step, automated, computer-assisted procedure. This tool enables the scientist to perform optimal elutions without requirement of prior knowledge about physical properties of the target molecule, and it presents instantaneous information on transfer efficiency. Furthermore, Selective RetrievalTM is a repeatable, non-destructive process. Multiple zones can be eluted sequentially. Selected multiple lanes can be eluted simultaneously. Eluted sample components from several lanes can be pooled to increase yields. The present example evaluates this automated process to yield pure, SDS-free BSA from a crude sample for its direct subjection to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. .

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[1/4-2]
Gombocz, E., Cortez, E., Yang, L. and Rammler, D.: New Method for Rapid High Efficiency Retrieval of Proteins for MALDI-TOF Mass Spectrometry, Using the HPGE 1000 System. J.Biol.Chem., submitted (1996).

A new approach is introduced for zone-selective, vertical electroelution of biologically intact, unlabeled proteins from a horizontal electrophoretic separation directly into small containers for mass spectrometric analysis. This method uses a fully automated gel electrophoresis system, avoiding difficulties common to blotting and electroelution of gel slices. Real-time fluorescence photometric determination of the transfer efficiency, the ability for immediate use of the eluate for confirming mass spectrometry without additional treatment, and the benefits of the method are demonstrated on Bovine Serum Albumin and Human Serum Albumin retrieval from a crude sample. The described high performance gel electrophoresis (HPGE) system consists of a computer-controlled instrument to perform electrophoretic protocols under active feedback loop control for all parameters influencing the separation. It allows for dynamic zone monitoring during the entire experiment to identify and extract any separated component under pre-set conditions at any time during the run. In 2 examples (crude BSA and HSA), SDS-free pure proteins were automatically eluted from the separation gel and directly subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Molecular weights obtained both by mass spectrometry and electrophoresis were compared, resulting in a relative deviation of 0.32% and 0.03%. Labeled and unlabeled samples were separated side-by-side on a 5%T MetaPhor Agarose gel in SDS at constant field strength prior to elution. FLUOS-labeled samples were used as a reference. Since the system allows for rebuffering during the automatic electroelution procedure, the recovered SDS-free unlabeled proteins were used without further purification. The pure proteins at concentrations of 650 pmol/`l(HSA) and 750 pmol/`l (BSA), were retrieved in only 2 minutes into a small volume of SDS-free buffer with efficiencies of 92.6% (HSA) and 95.8% (BSA) automatically in a single step. The Windows-based GLP-compliant operating software provides the user with mobility and migration information on each individual protein, thus accounting for high yield transfer efficiencies (typically between 92% - 98%) even on unknown proteins without the need for individual optimization. Qualitative, quantitative and graphical reports document all experimental conditions and results. The analytical and preparative aspects of this new method for rapid, gentle and selective protein transfer without loss of biological activity are discussed.

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[1/5]
Gombocz, E. and Cortez, E: Analytical and Preparative Impact of HPGE on Genomics and Proteonomics. Am.Biotechn.Lab. 15 (9), 66-72 (1997).

Genome and Proteonome scientists need quick, reliable, quantitative and reproducible tools. The ongoing merge between biochemistry and information science accounts for the fact, that scientists are more interested in structure, function and interaction of or between macromolecules. In Genomics, separation of ssDNA, dsDNA, RFLP's, PCR product charceterization and high speed chromosomal FIGE are required. Using HPGE, for the first time conformational changes during the separation and site-specific mutation in TTGE can be monitored in real-time. In Proteonomics, quantitation is extended to biological functionality by multi-color, real-time kinetics, using In-Gel Assays for enzymatic or immunological activity. A systems approach which considers molecular properties can support analytical and preparative needs simultaneously. High Performance Gel Electrophoresis (HPGE) as a fully auto- mated, computerized separation systems approach has refocused the importance of gel electrophoresis. The proven concept, paired with ease of use, auto- mation, real-time visualization and data management - particularly, since GLP/GMP compliance is required now in every laboratory - has lead to a significant impact of this technique on Genomics and Proteonomics. The ability to concentrate, separate, retrieve (elute) and assay in one system is the key to quick and insightful answers towards the complexity of functional connectivity and interactions between genes and peptides. On the advent of powerful new technologies, HPGE has gained momentum as method of choice to provide pure components for MS, sequencing and multidimensional tools as well as for fast, documented analytical screening in production, synthesis and quality control.

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[1/6]
Gombocz, E., Cortez, E. and Rammler, D.: HPGE: Concept of integrated automation and its applications. Electrophoresis 19,in press (1998).

High Performance Gel Electrophoresis (HPGE) as a fully automated, computerized separation systems approach has refocused the importance of gel electrophoresis. Single base separation of ssDNA and dsDNA, RFLP's or PCR products with single-step electroelution and high speed chromosomal FIGE and TTGE for site-mutation detection have demonstrated its potential in Genomics. In Proteonomics, quantitation is extended to biological functionality by real-time kinetics in-gel assays for enzymatic or immunological activity. Dynamically obtained, mobility-based molecular weights in HPGE agree with those of mass spectrometry and sequencing. Using semi-solid media, HPGE allows to characterize mitochondria's, cell particles and viruses within the same unit. Both, Genome and Proteonome scientists benefit from instant, non-destructive retrieval of pure components for MS or sequencing and the ability to reapply automatically for multi-dimensional techniques. Automated, on-line Ferguson plot analysis provides information on shape, folding/unfolding, aggregation or denaturation. Besides increased productivity, the parameter feedback in the HPGE environment has increased confidence in and reproducibility of results, leading to standardized, reliable data. Automation and ease of use provide the base for high-end, next step analysis tools which make gel electrophoresis again the method of choice to gain more insights on interactions and functions of molecules.

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[1/7]
Gombocz, E., Yefimov, St., Alfred L. Yergey, A.L. and Chrambach A.: Transfer of native protein from gel electrophoretic zones into mass spectrometry, using electroelution of the band into buffer without sectioning of the gel. Electrophoresis, submitted (1998).).

The automated gel electrophoresis apparatus HPGE-1000 comprises a unique preparative module in which an elution cup filled with buffer descends on the band of interest under computer control, allowing for direct electroelution of the analyte from the band into the buffer. This electroeluate buffer can then be subjected to mass spectrometry for identification of the analyte. Potentially, that procedure of transferring protein from the gel electrophoretic band to mass spectrometry without any gel staining and sectioning is superior to the present practice of diffusing proteins from gel slices. However, that potential cannot be realized unless the gel electrophoretic separation of the protein to be electroeluted can be carried out on fluorescently unlabeled proteins, since the mass spectrometric analysis cannot tolerate a derivatization of the protein with a fluorophore which would alter the mass. Thus, to date it has been applicable only i) in application to naturally fluorescing proteins, ii) in application to bands from gel lanes run in parallel with fluorescently labeled protein, correcting for the mobility difference between labeled and unlabeled protein, and iii) by detection of unlabeled protein using relatively insensitive "fluorescence reduction". Using i), it was possible to demonstrate that electroeluted naturally fluorescing recombinant Green Fluorescing Protein (rGFP) exhibited an identical mass in MS before and after electroelution, i.e. that it maintained its molecular integrity during gel electrophoresis and electroelution. The new and improved approach to the problem is based on Cascade Blue, which under described MBE conditions does not react with proteins covalently but was found to label native proteins (in the absence of SDS) in solution. Native alpha-lactalbumin could clearly be visualized through non-covalent association with the fluorophore. Similar data were obtained with soybean trypsin inhibitor, ovalbumin, serum albumin and conalbumin (Mr's of 22.7, 45, 67 and 86 kDa, respectively). Interference of Cascade Blue with mass spectrometric analysis of a-lactalbumin was tested by electroelution of the protein and analysis of an aliquot of the electroeluate on a PerSeptive BioSystems Voyager Elite DE-STR MALDI-TOF instrument. The presented data confirm that the original sample of a-lactalbumin is indistinguishable in mass from the electroeluate of the protein after electrophoresis in the presence of the dye in the gel. Tryptic peptides derived from original sample and electroeluate were also shown to be indistinguishable. The yield of a-lactalbumin in the electroeluate determined by protein assay, relative fluorescence intensity of the HPGE-patterns and the peak height in MS was 75%. It is concluded that electroelution of protein from gel bands visualized by Cascade Blue into buffer, using the HPGE-1000, followed by transfer to MS, presents a new and superior way of characterizing proteins by their masses.

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[1/8]
Gombocz, E. and Cortez, E.: Carrier Ampholoytes rehablitiated: Gel isolectric focusing on pH gradients visualized in real-time by automated fluorescence scanning in the HPGE-1000 apparatus. Electrophoresis, submitted (1998).

All synthetic carrier ampholyte mixtures (SCAMs) contain some naturally fluorescing carrier ampholytes (CAs). The detection of those during IEF, using gel electrophoresis apparatus with intermittent scanning of fluorescence, allows one to follow in real time the "life cycle" of the pH gradient, i.e. its genesis, steady-state and decay. The most prominently fluorescing CAs can be calibrated by pH measurement at or after the steady state ("calibration-CAs). By application of the calibration-CAs, the fluorescence pattern of CAs can be interpreted in terms of pH gradient (pIs). Simultaneously with the visualization of the pH gradient in that way, protein samples can be detected by "fluorescence reduction" and assigned pH values in dependence on focusing time and, at or after the steady state of the protein, pI'-values. The method remedies the inherent blindness of IEF with regard to the state of the pH gradient within its limited "life cycle". It allows one to load the sample at a time when the shape of the pH gradient is optimal for the purpose of its resolution from neighboring components. The visualization of the cathodic drift during IEF eliminates any of the danger to resolution and to loss of sample associated with "blind" IEF. Most importantly, the possibility to follow the pH in the position of the protein as a function of time provides an objective, accurate measure of the pI' not available from pH measurement at an arbitrary focusing time. The method therefore pre-empts the advantage of using an IEF method which is free of the pH gradient drift, i.e. IPG-IEF. Moreover, it preserves the "natural pH gradient", does not present any of its sample entry problems and those due to very low conductance, and is compatible with agarose gels and their relatively diminished restrictiveness to migration.

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[2/1]

[2/2]
Yarmola E, Chrambach A:Band width measurement in automated gel electrophoresis apparatus: DNA dispersion in a discontinuous system and in a single buffer. Electrophoresis 16 (3): 345-349 (1995).

Recent commercial introduction of automated gel electrophoresis apparatus allows for band width measurements during electrophoresis and therefore promises to open up the exploitation of band width and shape for the physical characterization of charged macromolecules in the same manner in which to date quantitative gel electrophoresis had exploited electrophoretic mobility at multiple gel concentrations. The measurements demonstrate decreased band width and therefore increased resolving power for a discontinuous buffer system compared to Tris-borate EDTA buffer. The dispersion coefficients (D' = (sigma 2-sigma 2o)/t) of homogeneous DNA components appear to decrease with gel concentration when either the field strength or the DNA length is small, and increase with gel concentration when these are large. This contrasting response of D' to increasing gel concentration is presumably due to DNA stretching, which increases in proportion to DNA length and field strength, and to the progressive orientation of agarose with increasing field strength.

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[2/3]
Yarmola E, Chrambach A: Nonlinear "Ferguson curves" by two runs of the commercial automated HPGE-1000 gel electrophoresis apparatus with intermittent scanning of fluorescence. Electrophoresis 16 (3): 350-353 (1995)

The exploitation of gel electrophoretic migration distance to gain information of molecular and gel fiber properties depends on the functions relating mobility with gel concentration. To the degree that these are nonlinear, the definition of those functions by past methods has been excessively laborious or, in application of gel concentration gradients, based on a number of assumptions. The recent commercial introduction of gel electrophoresis apparatus capable of intermittent scanning of the pattern promised to solve these problems. The present study shows that such apparatus allows for a precise definition of a nonlinear "Ferguson curve" (mobility vs. gel concentration) in two experiments, using different gel concentrations in the eight channels of the HPGE-1000 apparatus and 5-29 scans in each during the course of an electrophoretic run. Simultaneously, these Ferguson curves are obtained for five components of a DNA ladder ranging in DNA length from 121 to 1857 bp.

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[2/4]
Chrambach A., Yarmola E., Zakharov S.F., Garner M.M.: Commercial automated gel electrophoresis apparatus: application to DNA, band dispersion, nonlinear Ferguson curves, and isolation. Electrophoresis 16 (5): 713-718 (1995)

Recently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE-1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor-saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 micrograms load for a full-scale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching.

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[2/5]
Zakharov,S.F., Garner, M.M., and Chrambach, A.: Recovery of SDS-Proteins and DNA using commercial automated gel electrophoresis apparatus. Appl.Theor.Electrophoresis 5 (1): 25-29 (1995).

The HPGE-1000 apparatus (LabIntelligence, Menlo Park, CA) is a gel electrophoresis instrument with intermittent fluorescence scanning of the migration path and with preparative capability. An electroelution cup sealed with gel is placed onto the band of interest, identified and located under computer control, and the band is electroeleuted into the cup at a right angle to the orientation of the resolving gel. The correct location of the eluted band and the degree of its recovery into the elution cup are then verified on the gel pattern, visualized on the computer screen. Using that procedure, SDS-conalbumin-FLUOS was electrophoresed at 5 V/cm in a discontinuous tricinate-chloride-Tris system at loads of 0.25 to 20µg, using 5% agarose (MetaPhor, FMC), 0.03% SDS gel at 5°C. The horizontal gel was partitioned at the sample loading slit between a gel in Tris-tricinate (prepared at the concentration of an operative phase ZETA) and in Tris-chloride (prepared as phase BETA). The elution cup was sealed with latter gel and overlayered with buffer of the composition of the former. This arrangement should provide for electoeleution of the band as a highly concentrated stack. At electroelution times of 2, 3.5, 4-5, 15, and 15 min at 15 V/cm yields were 58, 60, 54-76, 99, 99 and 84% for loads of 0.25, 0.5, 1, 4, 10 and 20µg, respectively. At the most sensitive scale of detection (13), a full-scale peak was obtained at a load of 1.7 µg when the fluorophore (FLUOS, Boehringer-Mannheim) to protein ratio was 10:1. Similarly, homogeneous nucleosomal DNA (146 bp), electrophoresed in 0.2x TBE buffer at a load of 5µg, was near-quantitatively recovered into the same buffer by electroelution at 15 V/cm for 2.5 min or 6 min.

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[2/6]
Weiss, G.H., Garner. M.M., Yarmola, E., Bocek, P. and Chrambach, A.: A comparison of the resolution of DNA fragments between agarose gel and capillary zone electrophoresis in agarose solutions at 40°C. Electrophoresis 16: 1345-1353 (1995).

The resolving power of capillary zone electrophoresis (CZE) is compared to that of gel electrophoresis (GE) under similar conditions (agarose, similar length of DNA fragments, identical buffer) but with differences in temperature and field strength. The comparison is based on the time required to reach a desired degree of resolution by each of the two menthols. A resolution parameter is developed which is equally applicable to CZE, with relatively diffuse initial conditions in the absence of stacking and measurements expressed in terms of time, and to GE, in which measurements are expressed in terms of spatial parameters. The resolution time in CZE using agarose solutions at 40°C was found to be greater by at least on order of magnitude than that in GE using agarose gels. Thus, the increased migration velocity due to high field strength in CZE substantially outweighs the lower dispersion in GE.

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[2/7]

[2/8]
Chang HT, Chrambach A: Feasibility of electrophoresis of a subcellular-sized particle in polymer solutions, using automated horizontal gel apparatus. Appl. Theor. Electrophoresis 5 (2): 73-77 (1995) 

Electrophoresis in polymer solutions of a fluorescently labeled polystyrene carboxylate particle of 46.5 nm radius was carried out in a horizontal gel electrophoresis apparatus with intermittent scanning of the migration path. Polymers of the order of 10(6) (dextran, polyvinylpyrrolidone, polyacrylamide and polyethyleneglycol) and 10(5) (hydroxyethylcellulose, polyethyleneglycol) M(r) were used. In each application, bands formed, became symmetric and narrowed with increasing polymer concentration. The decrease in dispersion coefficients, D', with polymer concentration was sharpest with the polymers of M(r) 10(6); but significant differences in the effectiveness of polymers within that group also exist which are not accounted for. Provided that the demonstrated feasibility of banding in concentrated polymer solutions will allow for separations similar to those achieved at low polymer concentrations in CZE, the method promises to overcome the inherent shortcomings of capillary electrophoresis in polymer solutions with regard to inaccessibility of bands for immunological and affinity detection as well as to preparative scale and preparative instrumental complexity.

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[2/9]

[2/10]
Aldroubi A., Zakharov, S.F. and Chrambach, A.: A computer program for predicting recovery of SDS- protein in the automated HPGE-1000 apparatus. Appl.Theor.Electrophoresis 5 (1): 31-34 (1995).

The commercial automated gel electrophoresis apparatus (HPGE-1000 of LabIntelligence, Menlo Park, CA) allows one to recover the material migrating and visualized as a fluorescent labeled band by electrophoresis into a collection cup located above the band at right angle to the orientation of the separation path. The degree of recovery is a function of sample load (peak area), electrophoresis time at constant field strength, the mobility of the material and band width. Neglecting the latter, recovery of several SDS-proteins was measured as a function of the first three parameters. These measurements were used as a data base for computer program capable of predicting, by interpolation of the experimental values, the time of electrophoresis needed to obtain a specified degree of recovery, or the degree of recovery obtained after a desired time of electrophoresis into the collection cup.

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[2/25]
Lichtenwalter, K.G., Chakel, J.A., Apffel, A., Li, L. and Hancock, W.S.: The use of gel electrophoresis coupled with MALDI-TOF for the analysis of DNA in biological samples. HPLC'96, San Francisco (1996).

Known applications for MALDI-TOF in molecular biology are analyis of synthethic oligonucleotides and fluorescently tagged primer characterization. Objective of the present study was to determine, how the range of MS can be extended to allow analysis of "real" samples such as bacterial DNA. MALDI-TOF of several Oligonucleotides (Poly Thymidine, pBR322 plasmid sequences, 25 base pair (double stranded) extracted from gel and 21 base(single stranded) electroeluted from gel) and TLF (Time Lag Focusing) of 50 base oligonucleotide were performed to answer these questions. LC-ESI/MS applications demonstrated on a Poly-T mix was used to compare the achievable size range. LC/ESI-MS typically can be used for 1 to 50 bases, MALDI-TOF for 50 - 150 bases, and for larger DNA fragments after digestion. The matrix used in this study was 2,6-dihydoacetophenone mixed with dihydrogen ammonium citrate (1:1 with DHAP in ethanol). A 26 base pair DNA ladder and short oligonucleotides at 18, 19, 20, 21 and 22 bases were used for gel extraction and electroelution experiments. 25 nanogram of each short synthetic oligonucleotide was applied to a 4% GTG agarose gel in 1x TAE. Electrophoresis was performed at ~400 V, 34 mA, ~15°C for 40 minutes using an automated gel electrophoresis system (HPGE 1000A; LabIntelligence) capable of electroeluting DNA contained in selected gel zones. The oligonucleotides were electroeluted and collected in ~200 µl 1x TAE buffer (pH 8.4). The DNA was then precipitated, the precipitate collected by centrifugation at 14,000 x g, 30 minutes at 4°C, and the DNA was resuspended in 10 mL deionized water. In a comparison experiment, one microgram 25 base pair ladder was applied on a 3% GTG agarose gel in 1x TAE, and electrophoresis was performed conventionally at 110 V, 44 mA for 2 hours. The gel was stained with Ethidium Bromide and the 25 bp fragment was cut out of the gel. The DNA was extracted from the gel slice using GenElute Minus EtBr spin column centrifugation for 10 minutes at 14,000 x g. The DNA was precipitated and resuspended as described above. The experiments concluded, that MALDI-TOF is a valuable method for synthetic oligonucleotides. It can be used to get mass data on both, plasmid fragments and electroeluted oligonucleotides; however, better methods are needed for sample desalting and concentration. Time Lag Focusing appears very promising in extending the mass resolution and accuracy of MALDI-TOF. Specific cleavage methods are needed to fragment DNA (<100 bp).

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[2/47]
Chrambach, A. and Wheeler, D.L.: Capabilities and potentialities of transverse pore gradient gel electrophoresis. Electrophoresis 15 (8-9): 1021-1027 (1994).

Transverse pore gradient gel electrophoresis is important as a tool for obtaining nonlinear Ferguson plots [log(mobility) vs. gel concentration], e.g. in application to DNA in polyacrylamide gels or to agarose gels, with the purpose of evaluating molecular properties (size, conformation, malleability) and gel fiber properties (fiber radius and length per unit volume). To date, it is capable of (i) yielding gel patterns ("Ferguson curves") of migration distance vs. predicted % T-range of the pore gradient, assuming its linearity; (ii) yielding information regarding molecular conformation from the intersection of Ferguson curves of unknowns (e.g. bent DNA) with those of standards; (iii) acquisition of Ferguson curves by computer, using prototype instrumentation; (iv) mathematical manipulation of acquired Ferguson curves to yielding Ferguson plots, providing that mobility in free solution has been assessed by capillary zone electrophoresis. The potentialities of the method remain unfulfilled to date due to (i) the unavailability, with a single exception, of an accurate and precise way to produce pore gradients of known shape; (ii) unavailability of a routinely applicable analysis for % T; (iii) unavailability of optimized, user-friendly and foolproof instrumentation for computer acquisition of Ferguson curves, including the present inapplicability of a commercially available electrophoresis apparatus with intermittent optical detection to transverse pore gradient gels; and (iv) unresolved problems in the statistical evaluation of Ferguson curves.

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[3/1]
Gombocz, E. and Cortez, E.: Use of Multiple Short Gels for Elongated Separation Pattern, Using Real-Time Dynamic Information Management in a Computer Controlled System For Automated Analysis. Prod. Appl. Notes for HPGE System, PAN 1, LabIntelligence (1993).

In applications, where high resolution is essential and separation of a large number of sample components is the objective, elongated gel dimensions are in most cases the only way to account for these requirements. However, this approach buries significant drawbacks (e.g. extremely long run times, difficulties in thermostatization, zone broadening and diffusion of smaller sample components). The use of multiple short gels together with a computer controlled, fully automated real-time gel electrophoresis system capable of dynamic information management and allowing simultaneous, independent separations on different gel concentrations, remedy these obstacles. Since each of the lanes represents optimum sieving conditions for a particular size range, gels can be significantly shorter, require shorter run times, but still achieve high resolution with least diffusion and less zone distortion. Proprietary multi-step software modules allow for dynamic Ferguson plot calculation during the separation process and for calculation of cut-off points to generate an elongated separation pattern ("synthetic gel")without zone overlap. Applying this technique on agarose gels ranging between 0.5% T - 1.9%T, the usable size range for separation of DNA fragments extends over 80 bp - 2 Mbp, using a multiple of active separation path lengths of 120 mm (resulting in a calculated "synthetic gel" length of 640 mm). Real-time zone detection and dynamic calculation of data ensures fast, reliable, reproducible results of superior quality than those obtained from separations with long migration paths.

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[3/2]
Cortez, E. and Gombocz, E.: Concentration, Separation and Retrieval - A New Concept of Integrated Electrophoresis Automation, Demonstrated on Selected Examples for Proteins and DNA. Prod. Appl. Notes for HPGE System, PAN 2, LabIntelligence (1992).

A new concept of integrated electrophoresis automation is introduced, which includes electrophoretic procedures in several analytical steps: component selective concentration (step 1), separation with real-time dynamic parameter feedback and monitoring of separation dynamics in real-time(step 2) and selective retrieval of separated components of interest (step 3) in their native form, retaining their biological activity and allowing for re-application in multi-dimensional techniques. Computerized system automation ensures optimum reproducibilty by active control of all influence parameters (buffer conditions, temperature, fieldstrength across the gels, current) and allows for real-time detection of absorbance and fluorescence emission simultaneously. The capability for selective stacking prior to separation together with a uniquely designed retrieval system, which allows for selective pickup of separated sample components supports the user with a powerful analytical real-time instrumentation for discrete identification tests (sequencing, enzyme activity measurements, immuno- detection, dedicated visualization techniques), immobilization or re-application. Since zone migration is tracked during the entire run, components may be picked up at any time during the entire separation process, allowing for fast protocols to isolate and characterize sample components analytically or semi-preparatively without change of their original physical characteristics. The benefits of this technology are demonstrated on selected examples of different separations and isolations of relevant proteins and DNA fragments.

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[3/3]
Gombocz, E. and Cortez, E.: Automated restriction fragment length determination of dsDNA using an integrated real-time electrophoresis system. Prod. Appl. Notes for HPGE System, PAN 3, LabIntelligence (1993).

Restriction fragment length analysis is a widely used and important tool in many areas of molecular biology. Current drawbacks are long run times, labor-intense techniques and relatively poor reproducibility. To obtain high resolution reproducible DNA fragment separations, automation of the entire analytical process is the key for both, high throughput and high quality results. This paper describes a new concept of a computer controlled real-time system that fulfills these needs. The system performs the entire analytical process from sample application, separation, dynamic zone detection to the final evaluation of results in a fully automated fashion. During the entire experiment, all parameters influencing the separation are constantly monitored and controlled via active feedback loops. The built-in photometer allows for direct flourescence detection of the separation progress dynamics in the entire gel in real-time, thus avoiding labor-intense post-processing steps for visualization. The easy-to-use Windows-based software provides great flexibility in research while still maintaining simple push-button procedures for routine applications and providing an experimental database complying with GLP guidelines for forensic laboratories. Due to the systems concept, runs can be performed much faster (16 minutes field-inversion 24 V/cm effective field strength protocol for fragment sizes between 50 bp - 50 Kbp), standardized, reproducible and completely automated, resulting in accurate quantitation and size determination of fragments. The demonstrated separation comparison exemplifies the benefits of an universal automated real-time system and represents an example for effective laboratory automation of DNA restriction fragment length determination.

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[3/5]
Gombocz, E. and Cortez, E.: A New Way of Protein Transfer and Immobilisation: Automated, Zone-Selective Electroelution of Proteins from SDS Gels with Instantaneous Photometric Determination of Transfer Efficiency in an Integrated System. Prod. Appl. Notes for HPGE System, PAN 5, LabIntelligence (1994).

Blotting of proteins from gels, although widely used, currently is limited by three major obstacles: the fact, that transfer efficiency for different sized proteins will be significantly different due to compromising transfer conditions; that immediate information on transfer efficiency is not available; and, that the immobilizing medium may have adverse effect on the biological activity of the protein or its recovery thereof. This paper describes a complete new approach using an automated system, which allows zone-selective electroelution vertically into a small container with liquid buffer or gel plug directly off any horizontal electrophoretic separation pattern with photometric determination of the transfer efficiency in real-time. On examples of electroelution from HSA and BSA from crude extracts the computer-assisted retrieval process is described and evaluated. Since the system allows for rebuffering during the automatic electroelution procedure, the recovered SDS-free proteins were without further cleanup steps directly subjected to MALDI mass spectroscopy to confirm their purity. The Windows-based operating software provides the user with mobility and migration information on each individual protein, thus allowing for high yield transfer efficiencies (typically between 95% - 98%) even on unknown proteins without necessitating individual optimization. A discussion about analytical and preparative aspects of this new and fully automated approach for a rapid, gentle and selective protein transfer without loss of biological activity concludes the paper.

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[3/6]
Gombocz, E. and Cortez, E.: Molecular Weight Determination of Proteins in Real-Time: Quick Protocol Using Bovine Serum Albumin Oligomers as Size and Quantification Calibrators in a Computer Controlled Gel Electrophoresis System. Prod. Appl. Notes for HPGE System, PAN 7, LabIntelligence (1994).

Molecular weight determination of proteins, despite of its widespread use as a bioanalytical technique, still commonly lacks ease of use and accuracy. Automation of a generally applicable protocol with real-time evaluation of the separation pattern remedies these obstacles. The paper describes a quick and precise protocol for the automated HPGE 1000 High Performance Gel Electrophoresis System, which achieves accurate molecular weights in the range of 10 kD - 600 kD with a mobility precision of 0.5 rel% within 45-60 min. Auto- mated sample application and separated gel lanes improve the analytical quality of the experiment in high volume routine applications. Real-time zone migration is visualized via fluorescence photometry resulting in nano-gram sensitivity. The use of fluorescently labeled BSA oligomers as a homologuous protein family for quantification and size reference allows for easy and single-step wide-range calibration for both, concentration and molecular weight. The Windows based instrument software performs automatic peak search, segmentation, quantification and size determination based on true mobilities at any scan time during the experiment. GLP-compliant data storage, data retrieval and reporting makes the HPGE system attractive for molecular weight determinations in regulated, certified or forensic laboratories. Statistical data on lane-to-lane and run-to-run reproducibility for mobilities and peak areas using separation data obtained from serial dilutions of BSA oligomers on MetaPhor agarose gels, GTG agarose gels and polyacrylamide illustrate the reproducibility of the system. The advantages of real-time separation histories for data comparison are discussed.

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[3/7]
Gombocz, E. and Cortez, E.: Visualization of pH-Gradient Formation in Carrier Ampholyte Isoelectrofocusing: Gel Stabilization Dynamics Observed via Real- Time Photometry in an Automated Gel Electrophoresis System. Prod. Appl. Notes for HPGE System, PAN 8, LabIntelligence (1994).

Isoelectrofocusing in different varieties allows for characterization of charge isomers with extreme high resolution. While Carrier Ampholyte based isoelectrofocusing is a two step process (step 1: establishment of pH gradient, step 2: focusing of sample components), the completion of the first separation step typically relies on experience of the user. This paper focuses on an objective criteria for establishing the desired pH gradient prior to sample application without the use of gel additives. On examples of wide and mid-range carrier ampholyte mixtures, gel stabilization dynamics were recorded, using the automated HPGE 1000 High Performance Gel Electrophoresis System to visualize pH gradient formation in real-time via fluorescence photometry. Since most of the SCAM's exhibit significant intrinsic fluorescence when used in concentrations as typically applied in standard Agarose or Polyacrylamide isolectrofocusing protocols, direct visualization of the dynamics during pre-focusing of the gel is possible. The paper describes the influence of incomplete pH gradient establishment on band resolution using a set of defined IEF markers applied at different times and at different positions on the gel. Unlabeled, native protein zones were visualized as dark bands (fluorescence quenching) during their migration to their iso-pH's and their steady-state behaviour as well as the cathodic drift of the gradient documented. The technique also demonstrates the advantage of a photometric pH calibration for the gradient in its application to isoelectric point determinations and the benefits of a fully automated, computer controlled gel electrophoresis system in isoelectrofocusing.

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[3/18]
Gombocz, E.: Automated Nucleic Acid Separations with Atto-Mol Sensitivity and Real-Time Separation Dynamics via Fluorescence Photometry. Prod. Appl. Notes for HPGE System, PAN 18, LabIntelligence (1995).

Electrophoretic characterization of PCR-amplified DNA's and restriction fragment length analysis are widely used important tools in many areas of molecular biology. Current drawbacks are long run times, labor-intense techniques and relatively poor reproducibility. To obtain high resolution, reproducible DNA fragment separations, automation of the entire analytical process is the key for both, high throughput and high quality results. This paper describes a new concept of a computer controlled real-time system that fulfills these needs and allows in addition for separation dynamics monitoring with atto-mol sensitivity. The entire analytical process from sample application, separation, dynamic zone detection to the final evaluation of results is carried out in a fully automated fashion. During the entire experiment, all parameters influencing the separation including temperature and buffer conditions are constantly monitored and controlled via active feedback loops. The built-in photometer allows for direct fluorescence detection of the separation progress dynamics on the entire gel in real-time, capable of using multi-color labeling covering an emission range between 410 - 720 nm. Differently cross-linked Polyacrylamide and high resolution Agarose gels such as MetaPhor over a wide range of %T account for single base resolution as required for sequencing applications, resulting in both, accurate size determination and quantification of DNA fragments. Easy-to-use Windows-based system software provides great flexibility in research while still maintaining simple push-button procedures for routine applications and providing an experimental database complying with GLP guidelines for forensic or certified laboratories. The in-gel separation dynamics allows for real-time site-mutation detection in combination with temperature gradients and/or monitoring of conformational changes during the separation process, using Ferguson plot analysis. The separation patterns shown in this paper exemplify the possibilities and benefits of an universal, automated real-time system and represent an example on effective laboratory automation for a variety of different DNA applications.

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