bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐dimensional motion of DNA bands during 120° pulsed‐field gel electrophoresis
Authors: Neitzey Lynn M. , Hutson M. Shane , Holzwarth George
Published: May 2005

The position and velocity of a band of double‐stranded, linear DNA from bacteriophage G were measured during 120° pulsed‐field gel electrophoresis, using a video micrometer. Both the x and y coordinates were determined simultaneously in the plane of a 1% agarose gel; x is the mean drift direction. For pulse durations T greater than the tube renewal time T*, the path traced by the band of 670 kb DNA in the xy plane was in remarkably good accord with that predicted by Southern's ratchet model. However, the measured instantaneous velocity vx showed a sharp backward spike each time the field changed direction, with amplitude about twice the mean drift velocity. This spike is not consistent with models which assume a constant curvilinear velocity of DNA in a tube, nor with the biased reptation model without fluctuations. The corresponding measurements of vy showed a sharp positive spike with amplitude more than 3 times the plateau velocity in the y direction; neither model predicted this. The sharp velocity spikes are consistent with the idea that, for T> T*, a large fraction of the DNA chains are stretched into U‐shaped or herniated configurations. When the field changes direction, the arms of the U's and the hernias recoil rapidly in response to intramolecular DNA chain tension. Because the base of a U or hernia is fixed by gel fibers, the center of mass of the chain recoils backward every time the field changes direction.
Effect of one‐dimensional pulsed‐field gel electrophoresis on linear and circular DNA
Authors: Turmel Chantal , Noolandi Jaan
Published: May 2005

A summary of the three main one‐dimensional pulsed‐field strategies (zero‐integrated field, forward‐biased field, and high frequency modulation) used for separating DNA molecules without band inversion within a preselected size range is given. Each of these strategies has size‐specific features which make separations up to 6 Mbp possible. We applied the same methodology to circular DNAs varying in size from 2 kbp to about 4 Mbp. The migration of intermediate‐sized circular plasmids (50 kbp–400 kbp) under these pulse conditions remains unexplained. On the other hand, preliminary results show that the migration of very large molecules, which are expected to be circular, comigrate with linear chromosomes of the same size under certain pulse conditions. We hypothesize that sample preparation, or the effect of the pulsed field, can create breakage and linearize very large circular DNAs, or that very large circular DNAs (> 2 Mbp) act like linear DNAs of the same size when submitted to one‐dimensional pulsed‐field gel electrophoresis conditions. The most likely possibility is that some of the circular DNAs have been linearized with one break during sample preparation, giving rise to a band at about 4 Mbp. The circular DNAs with more than one break may form an indistin‐guishable smear.
High‐performance capillary electrophoresis of glycoconjugates
Authors: Novotny Milos V. , Sudor Jan
Published: May 2005

Capillary electrophoresis (CE) has recently emerged as a highly promising technique for the analysis and structural study of glycoconjugates. This article reviews the current status of CE and its potential in glycobiology. The separation media suitable for the analysis of monosaccharides, oligosaccharides and very large carbohydrate‐based biomolecules are discussed. The detection aspects emphasize chemical derivatization approaches to tagging the glycoconjugates of interest for enhanced response in absorption and fluorescence spectroscopy.
Capillary zone electrophoresis of biological substances with fused silica capillaries having zero or constant electroosmotic flow
Authors: Smith Joel T. , Rassi Ziad El
Published: May 2005

A series of capillary surface modifications entailing multilayered coatings were introduced and evaluated in capillary zone electrophoresis of biological substances, e.g., proteins, peptides, oligosaccharides and nucleotides. In one set of surface modifications, large molecular weight hydroxypropyl cellulose afforded “zero” flow capillaries, which were used as precursors for developing anodal flow capillaries. When “zero” flow capillaries were further functionalized with a charged polyethyleneimine layer to which a top polyether layer was covalently attached, the resulting anodal electroosmotic flow was relatively weak due to the high viscosity of the coated wall imparted by the hydroxypropyl cellulose layer. Capillaries with relatively strong and constant anodal electroosmotic flow were best achieved when the inner capillary surface was first chemically derivatized with methylated (i.e., quaternarized) polyethyleneimine hydroxyethylated. This hydroxylated and permanently charged polymeric coating yielded constant anodal flow regardless of electrolyte pH. The hydroxyl groups of the charged polymeric coating permitted the covalent attachment of polyether chains, which minimized electrostatic interaction between the positive charges of the polymeric layer and oppositely charged biopolymers. Under these conditions, rapid transport of acidic biopolymers past the detection point in the separation capillary could be achieved, and relatively high plate counts were obtained.
Application of a diode‐array detector in capillary electrophoresis
Authors: Beck Wolfgang , van Hoek Roger , Engelhardt Heinz
Published: May 2005

In the last decade diode‐array detection has proved to be extremely useful in high performance liquid chromatography in recording UV‐visible spectra directly and on‐line in the column effluent. In capillary electrophoresis (CE) only fast‐scanning detectors with long scan times (up to 2 s) are commercially available. A home‐made CE instrument with a diode‐array detector (DAD) was built and compared with a commercial fast‐scanning detector. It is shown that the fast‐scanning detector is advantageous when less than eight wavelengths are monitored. Due to the high data collection rate the DAD is superior if high resolution UV‐visible spectra are acquired or if several spectra of a narrow peak (usual peak width in CE below 5 s) are needed for peak purity control. It is shown that DAD detection can help to develop separation methods, to identify peaks according to their UV‐visible spectra, and to check peak purity.
A three‐dimensional plot for the display of transverse pore gradient gel electrophoresis data: Application to a kinetoplast DNA fragment of planar circular conformation
Authors: Wheeler David , Chrambach Andreas
Published: May 2005

A 219 bp fragment of kinetoplast DNA from Crithidia fasciculata has previously been shown to exhibit a gel concentration dependent, anomalously low migration rate in 3–10% polyacrylamide transverse pore gradient gels relative to that of straight standards. The anomaly was interpreted in accordance with previous results of Marini et al. [1] as being due to the planar circular conformation of the fragment. The transverse pore gradient gel pattern is displayed here as a three‐dimensional plot of migration distance vs. gel concentration and DNA length for the kinetoplast fragment and straight DNA standards. The plot yields a smooth surface for the standards clearly separated from the curve of the kinetoplast DNA fragment. Thus, transverse gradient gel electrophoresis in conjunction with the three‐dimensional plot provides a unique method for the recognition of abnormal conformational features of DNA on the basis of electrophoretic mobility in a pore‐size gradient.
An electrophoretic study of the thermal‐ and reductant‐dependent aggregation of the 27 kDa component of ammonia monooxygenase from Nitrosomonas europaea
Authors: Hyman Michael R. , Arp Daniel J.
Published: May 2005

Standard protocols for sample preparation for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) typically involve the combined use of heat and a reductant to fully disrupt protein‐protein interactions and allow for constant ratios of SDS‐binding to individual polypeptides. However, 14C‐labeled forms of the membrane‐bound, active‐site‐containing 27 kDa polypeptide of ammonia monooxygenase from Nitrosomonas europaea undergo an aggregation reaction when cells or membranes are heated in the presence of SDS‐PAGE sample buffer. The aggregate produced after heating at 100°C is a soluble complex which fails to enter the stacking gel in discontinuous SDS‐PAGE gels. The extent of the aggregation reaction is dependent on the temperature of sample preparation, and the reaction exhibits first‐order kinetics at 65°C and 100°C (rates constants = 0.07 and 0.35 min−1, respectively). The rate of the aggregation reaction is further dependent on the concentration of reductant used in the sample buffer. However, the concentration of SDS does not significantly affect the rate of aggregation. The aggregated form of the 27 kDA polypeptide can be isolated by gel‐permeation chromatography in the presence of SDS. The aggregated protein can also be returned to the monomeric state by incubation at high pH in the presence of SDS. The aggregation reaction also occurs with 14C2H2‐labeled polypeptides in other species of autotrophic nitrifiers and a methanotrophic bacterium which expresses the particulate form of methane monooxygenase. We conclude that strongly hydrophobic amino acid sequences present in ammonia monooxygenase are responsible for the aggregation phenomenon.
Detection of neurotensin in tissues by capillary zone electrophoresis
Authors: Denoroy Luc
Published: May 2005

An electrophoretic method for detecting neurotensin in tissues was developed. Homogenates of rat duodenum and adrenal glands were first extracted by solid‐phase extraction and purified by reversed‐phase high‐performance liquid chromatography. The neurotensin‐rich fraction was further analyzed by capillary zone electrophoresis (CZE) using a commercial instrument with UV detection. A minor peak was detected as neurotensin and its identity was further confirmed by performing several CZE analyses at different pH values. Such an approach could be used to analyze with good molecular specificity the neuropeptides in some human tissues.
Factors affecting the determination of drugs and endogenous low molecular mass compounds in human serum by micellar electrokinetic capillary chromatography with direct sample injection
Authors: Schmutz André , Thormann Wolfgang
Published: May 2005

Factors influencing the establishment of an analytical window in front of the solubilized proteins in micellar electrokinetic capïllary chromatography (MECC) with direct serum injection (DSI) are discussed. Both drugs and endogenous low molecular mass compounds eluting within the analytical window are identified concurrently by multi‐wavelength absorption detection. Variables such as the concentration of the micelle forming substance, ionic strength, applied voltage, initial sample zone length, capillary length, selected buffer additives, insufficient renewal of the buffer in the anodic buffer vial and sample matrix are shown to impact MECC of endogenous compounds and model drugs, such as antiepileptics. For two drugs eluting within the analytical window, phenobarbital and ethosuximide, serum levels determined by DSI with external calibration are shown to compare well with levels obtained after liquid‐liquid extraction and internal calibration (use of an internal standard). In addition, reproducibility of both assays is excellent. The limit of employing DSI is demonstrated with the determination of the hydrophobic drug phenytoin. Using an automated, commercial instrument and naproxen as model drug, high‐speed MECC separations of high reproducibility and with a throughput of 12–15 samples per h are presented.
A theoretical approach to chiral capillary electrophoresis with some practical implications
Authors: Wren Stephen A. C. , Rowe Raymond C. , Payne Robin S.
Published: May 2005

A simple model has been proposed which explains enantiomeric resolution in capillary electrophoresis in terms of differential interactions with a chiral selector and differential migration of the two enantiomer‐chiral selector complexes. The equations derived allow predictions to be made and there is good support for the model in literature data.
Enantiomer separation using mobile and immobile cyclodextrin derivatives with electromigration
Authors: Mayer Sabine , Schurig Volker
Published: May 2005

The principle of electrochromatography is applied to enantiomer separation with wall‐coated capillaries of 50μm (ID). As chiral stationary phase an immobilized polysiloxane containing chemically bonded monokis‐6‐O‐octamethylene‐permethyl‐cyclodextrin (β‐ or γ‐Chirasil‐Dex) was used. The results are compared with the conventional method of adding cyclodextrin derivatives as chiral pseudostationary phase to the running buffer.
Separation of enantiomers by affinity electrokinetic chromatography using avidin
Authors: Tanaka Yoshihide , Matsubara Norio , Terave Shigeru
Published: May 2005

Avidin, a basic protein isolated from egg white, was employed as a chiral selector in affinity electrokinetic chromatography (EKC) for the separation of acidic enantiomers. Optical isomers of vanilmandelic acid, warfarin, ibuprofen, ketoprofen, flurbiprofen, and folinic acid were successfully resolved within 20 min with 25 μM avidin in a linear polyacrylamide‐coated capillary under weakly acidic conditions. The effects of pH, the concentration of avidin, addition of an organic solvent such as ethanol and 2‐propanol, and temperature on enantioselectivity were investigated. The choice of pH and the organic solvent additive was critical to improve the separation. Some general considerations about affinity EKC are also described, based on a simple one‐site interaction model.
Fluorescent‐based typing of the two short tandem repeat loci HUMTH01 and HUMACTBP2: Reproducibility of size measurements and genetic variation in the swedish population
Authors: Holgersson Stig , Karlsson Jan‐åke , Kihlgren Anne , Rosén Björn , Savolainen Peter , Gyllensten Ulf
Published: May 2005

The aim of this study was to investigate the reproducibility of genetic typing of two tetrameric short tandem repeat (STR) loci and the extent of genetic variation in the Swedish population. An automated, fluorescent‐based Applied Biosystems 373A sequencer was used for typing of the HUMTH01 and HUMACTBP2 loci (also named SE33). The former locus has seven alleles in the size range of 154–174 bp, while the latter is a complex locus with more than 32 alleles in the range of 227–316 bp. Using different fluorescent dyes, polymerase chain reaction (PCR) products from the two STR loci were sized in one lane using an internal size standard. In order to compare within‐ and between‐gel reproducibility of fragment size estimates, a control sample was typed three times on each of 20 gels. Within the gel, the standard deviation (SD) of fragment size variability was less than 0.1 bp for four fragment sizes between 158–291 bp. Standard deviations between gels were slightly higher for the two shorter fragment sizes (HUMTH01), while the larger fragments varied between 0.3 and 0.4 bp (HUMACTBP2). The amount of genetic variation was investigated in samples from three Swedish cities (n = 301). Seven alleles were found at HUMTH01 and the observed heterozygosity was 0.77. At the HUMACTBP2 locus more than thirty alleles were found and the observed heterozygosity was 0.96. The observed genotype frequencies at HUMTH01 and HUMACTBP2 did not deviate significantly from Hardy‐Weinberg expectations. No indication of a significant excess of homozygotes was found at any of the loci. We conclude that both HUMTH01 and HUMACTBP2 can be reliably typed using the method described. However, the latter locus requires an allelic ladder to be run on each gel.
The use of polyacrylamide gel electrophoresis for the analysis of acidic glycans labeled with the fluorophore 2‐aminoacridone
Authors: Jackson Peter , Pluskal Malcolm G. , Skea William
Published: May 2005

A method is described for the electrophoretic analysis of fluorophore‐labeled acidic glycans. Various glycoproteins were treated with the enzyme, peptide N‐glycosidase F, to release mixtures of asparagine‐linked glycans that were then labeled with the fluorophore 2‐aminoacridone, and the resulting derivatives electrophoresed in high‐density polyacrylamide gels. The acidic glycans were separated with high resolution and distinct, well‐resolved fluorescent band patterns were produced. Neutral saccharides did not electrophorese in the system. Information on the structure of the glycans was revealed by the changes in the band patterns observed after the mixtures of the glycan fluorophore derivatives were treated with either of the enzymes neuraminidase or β‐galactosidase. Quantities of glycoprotein as little as 10 μg were analysed without difficulty. The electrofluorograms were viewed using a digital imaging system based on a cooled charge‐coupled device. The method was also demonstrated using purified acidic glycans of known structure. The method was easy to use, enabled the sensitive analysis of multiple samples in parallel and should be a useful addition to the range of glycan analytical techniques.
Preparative isoelectric focusing in multicompartment electrolyzers: Novel, hydrolytically stable and hydrophilic isoelectric membranes
Authors: Chiari Marcella , Nesi Marina , Roncada Paola , Righetti Pier Giorgio
Published: May 2005

Preparative isoelectric focusing in multicompartment electrolyzers is based on the production of isoelectric membranes of precise isoelectric point, able to buffer at their pI value and to titrate proteins tangent to or crossing the membranes. Up to the present, such membranes have been based on polyacrylamide chemistry; acrylamide, however, is neither stable in acidic nor basic environments. We describe here novel membranes, produced with a unique monomer, N‐acryloylaminoethoxyethanol (AAEE). Poly(AAEE) membranes are extremely stable to alkaline hydrolysis (500 times more stable than polyacrylamide) and even more hydrophilic than the latter matrix. This allows production of highly reproducible membranes (these do not change their pI with time, since no acrylic acid is produced by hydrolysis upon storage) which do not adsorb proteins by hydrophobic interaction.
Capabilities and potentialities of transverse pore gradient gel electrophoresis
Authors: Chrambach Andreas , Wheeler David L.
Published: May 2005

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) unvailability 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.
Electrophoretic analysis of seed storage proteins from gymnosperms
Authors: Allona Isabel , Collada Carmen , Casado Rosa , Aragoncillo Cipriano
Published: May 2005

Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), pore gradient gel electrophoresis (PGGE) followed by SDS‐PAGE and Western blot analysis were used to characterize the seed storage proteins from seven gymnosperm species from the families Pinaceae (Abies alba, Cedrus atlantica and Picea abies), Cupressaceae (Biota orientalis, Chamaecyparis lawsoniana and Cupressus arizonica), and Taxaceae (Taxus baccata). SDS‐PAGE and PGGE X SDS‐PAGE indicate the presence of proteins with characteristics similar to the 7S globulins in all the species studied. Antibodies to a 7S globulin subunit from Pinus pinaster cross‐reacted with homologous polypeptides from Pinaceae species, but not with corresponding subunits from species belonging to other families. Also detected in each of the studied species, with the exception of A. alba and T. baccata, were proteins potentially homologous with 11S proteins. Of these proteins, only those of C. atlantica may be extracted by saline buffer, while the remainder require a dissociating agent. Antibodies raised against the small subunit from P. pinaster 11S protein recognized only the corresponding polypeptides from Pinaceae species. Overall, these results help clarify our knowledge of gymnosperm seed storage proteins.
Differential splicing of the glycophorin A mRNA
Authors: Omi Toshinori , Kajii Eiji , Iwamoto Sadahiko , Tsuchida Shuichi , Ikemoto Shigenori
Published: May 2005

Two truncated mRNAs, in contrast to the full length of mRNA associated with the glycophorin A gene (GpA‐TI, GpA‐TII), were isolated from erythroid cells cultured by the selective two‐phase liquid culture system for erythroid progenitors in peripheral blood from a normal individual. The GpA‐TI mRNAs displayed a direct transition from exon I to exon III, so that the deletion of exon II resulted in the deletion of 33 amino acids encoded by this exon. Furthermore, the GpA‐TII showed two direct transitions from exon I to exon III and from exon III to the exon V of the GpA gene. This mRNA lacked both exons II and VI, resulting in the deletion of 46 amino acids. Is is concluded that these truncated mRNAs are transcribed from the same gene as the GpA gene and correspond to splicing isoforms lacking different exons.
Molecular modeling of acrylamide derivatives: The case of N‐acryloylaminoethoxyethanol versus acrylamide and trisacryl
Authors: Miertus Stanislav , Righetti Pier Giorgio , Chiari Marcella
Published: May 2005

Molecular mechanics (for evaluation of total energies of individual structures of monomers and oligomers) and molecular dynamics (for evaluating dynamic dependencies of structural features) were used for obtaining information on some unique chemical behavior of a novel N‐substituted acrylamide (N‐acryloylaminoethoxyethanol; AAEE) vs. conventional acrylamide and trisacryl (N‐acryloyl‐2‐amino‐2‐hydroxymethyl‐1, 3‐propane diol, an extremely hydrophilic derivative; Tris‐A). As free monomers, Tris‐A degrades with zero‐order and acrylamide with first‐order kinetics, whereas AAEE is highly resistant to hydrolysis. It is found that Tris‐A (and its dideoxy derivative) is constantly forming hydrogen bonds between the OH groups and the carbonyl of the amido group (bond distances of 1.64 to 1.70 Å); this activates a mechanism of “NO acyl transfer” which leads to quick degradation of the amido bond even under mildly alkaline conditions. Conversely, AAEE (which also contains an ω‐OH group increasing its hydrophilicity) has no tendency to form H‐bonds with the amido carbonyl, thus being resistant to the above degradation mechanism. In fact, the oxygen in the ethoxy moiety of the N‐substituent chain acts as a preferential partner for H‐bond formation with the ω‐OH group. In the oligomeric state, it is found that Tris‐A (tetrameric and dodecameric structures were simulated) tends to form inter‐residue H‐bonds (approximately parallel to the growing chain) competing with the intra‐residue H‐bonds (folding onto the amido carbonyl and approximately perpendicular to the oligomer chain), thus greatly increasing its stability. However, none of these polymeric structures can compete with a poly(AAEE) matrix, which still shows a 500‐fold greater resistance to alkaline hydrolysis.
Fractionation of human serum proteins by capillary and recycling isotachophoresis
Authors: Caslavska Jitka , Gebauer Petr , Thormann Wolfgang
Published: May 2005

The fractionation of human serum proteins using capillary isotachophoresis (CITP) and recycling isotachophoresis (RITP) in presence of low molecular mass spacer compounds is reported. Anionic CITP was performed in an instrument equipped with a Teflon capillary of 0.5 mm ID as well as in an apparatus which features an open‐tubular fused‐silica capillary of 75 μm ID. RITP was performed in a recycling fast flow focusing apparatus in which fluid flows rapidly through a narrow, rectangular cell and the effluent from each outlet port is reinjected into the electrophoresis chamber through the corresponding input port. Typically, 1 mL serum was processed batchwise within about 2.5 h prior to collection of 30 fractions of about 4 mL each. The fractions were analyzed separately for conductivity, pH and UV absorbance and selected fractions were characterized by an immunoassay for transferrin, as well as by gel isoelectric focusing, two‐dimensional gel electrophoresis, CITP and capillary zone electrophoresis. The search for suitable electrolyte systems and spacers was executed by CITP and by computer simulation. For simple configurations, separations predicted by simulation are shown to qualitatively agree with fractionation performed by CITP and RITP. Configurations producing three protein subgroups, the first containing mainly albumin, the second transferrin and the third the globulins, are discussed.
The use of protein synthesis elongation factor EF‐Tu as internal calibration standard in two‐dimensional electrophoretic studies of differentiation in Streptomyces
Authors: Xin ‐ming Li , Vohradský Jiři , Weiser Jaroslav
Published: May 2005

Protein synthesis elongation factor EF‐Tu is presented as an internal calibration standard for quantitative analysis of two‐dimensional (2‐D) protein electrophoresis gels. EF‐Tu was selected on the basis of concentration measurements in cell‐free extracts from Streptomyces aureofaciens, grown under conditions leading to production of tetracyclines, and separated on one‐dimensional (1‐D) and 2‐D electrophoresis gels. The results demonstrated that the amount of EF‐Tu synthesised in S. aureofaciens under conditions of slow growth during production of tetracyclines is constant in proportion to all other de novo synthesized proteins regardless of their total number. This makes EF‐Tu an ideal internal protein standard for quantitation of protein spots on 2‐D electrophoresis gels. For such quantitative analysis we developed a computer‐aided image analysis system which provides preparation of a gel image for further analysis including calibration, background subtraction and cleaning for streaking in both directions. The system can locate any resolvable spot in the gel and measure the integrated density of the spot, even in the case of irregular spot shape in crowded and overlapping spot regions.
Computer simulation of pulsed field gel runs allows the quantitation of radiation‐induced double‐strand breaks in yeast
Authors: Kraxenberger Alfred , Friedl Anna A. , Kellerer Albrecht M.
Published: May 2005

A procedure for the quantification of double‐strand breaks in yeast is presented that utilizes pulsed field gel electrophoresis (PFGE) and a comparison of the observed DNA mass distribution in the gel lanes with calculated distributions. Calculation of profiles is performed as follows. If double‐strand breaks are produced by sparsely ionizing radiation, one can assume that they are distributed randomly in the genome, and the resulting DNA mass distribution in molecular length can be predicted by means of a random breakage model. The input data for the computation of molecular length profiles are the breakage frequency per unit length, α, as adjustable parameter, and the molecular lengths of the intact chromosomes. The obtained DNA mass distributions in molecular length must then be transformed into distributions of DNA mass in migration distance. This requires a calibration of molecular length vs. migration distance that is specific for the gel lane in question. The computed profiles are then folded with a Lorentz distribution with adjusted spread parameter Γ to account for and broadening. The DNA profiles are calculated for different breakage frequencies α and for different values of Γ, and the parameters resulting in the best fit of the calculated to the observed profile are determined.
Focusing of alkaline proteases (subtilisins) in pH 10–12 immobilized gradients
Authors: Bossi Alessandra , Righetti Pier Giorgio , Vecchio Giuseppe , Severinsen Steen
Published: May 2005

Isoelectric focusing in very alkaline immobilized pH gradients (IPG) was adopted for checking the purity and assessing the pI value of two strongly alkaline proteases: Savinase and Durazym. The first enzyme (known to be the most alkaline) contains 5 Asp, 5 Glu, 7 His, 7 Tyr, 5 Lys, no Cys and 8 Arg residues and should have a theoretical pI of 9.7. Yet, when focused in a pH 9–11 IPG interval, it was lost in the cathodic compartment. After repeated attempts at creating even more alkaline pH intervals, a pH 10–12 IPG range was finally optimized and proved successful in focusing both enzymes midway between the two electrodic compartments. The pI of Savinase was measured as 11.15 ± as 0.15; that of Durazym as 10.95 ± 0.20 and that of the pI marker cytochrome c as 10.6 ± 0.17. Both enzymes (and a number of minor components in each preparation) were proven to be active by an in situ zymogram consisting of a casein/agar overlay. The discrepancy between theoretical and experimental pI values could not be fully reconciled: when correcting for pK values of amino acids in proteins at 10°C, instead of the tabulated values at 25°C, the pI should increase to a value of 10. Differential UV spectra showed that ca. 1/2 Tyr are buried in the protein interior and are thus unable to contribute to surface charge. This further increases the pI value by 0.3 pH units to a pI of 10.3, still quite removed from the experimentally assessed pI value (in the gel, at 10°C) of 11.15.
Sample application by in‐gel rehydration improves the resolution of two‐dimensional electrophoresis with immobilized pH gradients in the first dimension
Authors: Rabilloud Thierry , Valette Cecyl , Lawrence Jean Jacques
Published: May 2005

We describe a modification in the sample application mode for isoelectric focusing with immobilized pH gradients. Instead of being applied at the surface of the gel in a sample cup, the sample is introduced into the gel during the immobilized pH gradient strip rehydration step. This modification implies the use of low percentage gels (below 3.5%T) and specially designed, but simple, rehydration chambers. The main advantages are a uniform resolution without side effects and the possibility of handling large sample volumes (500 μL for a standard 3 × 160 × 0.5 mm strip), allowing micropreparative work (milligram samples) with a simple experimental design.
Restriction fragment length polymorphism: Molecular weight analysis and calculation with a scanner‐based computer system
Authors: Luckenbach Christine , Luckenbach Alexander , Ritter Horst
Published: May 2005

For an exact and reproducible molecular weight calculation, an essential requirement in all applications of DNA profiling, we present a new system, called RFLP‐MAC (restriction fragment length polymorphism – molecular weight analysis and calculation). The system is composed of a sensitive digital gray‐scale scanner unit and an Apple Macintosh computer supported by a powerful combination of a shareware and self‐developed software package. Molecular weights are calculated by profiling the optical density and band particle processing parameters to identify the unknown DNA fragments. Mathematical models were used to interpolate the transverse and horizontal migration directions of the molecular weight standards, insuring consistent results from gel to gel. All data are stored in a relational multi‐user database system. Descriptive statistics are performed and allele frequencies are analyzed. Procedures for image‐analysis, molecular weight calculation, statistical evaluations and database management were developed and run within the multitasking environment of 4th Dimension. Two sets of data, intragel and intergel measurements, are investigated in order to check the precision of the RFLP‐MAC system. All these data are subjected to a standard analysis of variance resulting in a maximal 3 SD value of 0.005 (0.288%) for the intragel and of 0.091 (1.654%) for the intergel variation.
Multilocus DNA fingerprinting: The independence problem in quantitative paternity testing
Authors: Krawczak Michael
Published: May 2005

A simulation study was performed in order to determine whether or not the assumption of independence, made in the quantitative analysis of multilocus DNA fingerprints, represents an inadmissible over‐simplification. A total of 10000 cases of true and false paternity, respectively, were simulated in twenty replicas of various genetic models. Log‐likelihood ratios (paternity vs. nonpaternity; LR) were calculated using published likelihood formulae and assuming position‐wise independence. The resulting LR distributions were compared to (i) the results of a classical analysis of the underlying genotype data, and (ii) the distributions expected from the likelihood model employed in the LR calculations. Although considerable discrepancies were observed between these distributions, decision making about paternity appeared to be only marginally affected, especially when only a fraction of each multilocus DNA fingerprint was analyzed.
Poly(ethylene glycol)methacrylate‐acrylamide copolymer media for hydrophobic protein and low temperature electrophoresis
Authors: Harrington Michael G. , Zewert Thomas E.
Published: May 2005

The properties of poly(ethylene glycol)methacrylate‐acrylamide copolymer media (PEGMACs) were exploited in two ways: (i) in the first‐dimensional gel of two‐dimensional electrophoresis (2‐DE) of hydrophobic proteins and (ii) for high speed, high‐resolution electrophoresis at low temperatures. In the first application, improved resolution and yield of isoelectric focusing (IEF) separations for the hydrophobic protein zein was achieved compared to IEF in standard polyacrylamide gels. This appears promising as a candidate approach for higher resolution 2‐DE mapping of uncharacterized hydrophobic proteins. In the second application, PEGMACs compatible with hydroorganic antifreeze buffer systems bellow cooling of the gels to low temperatures (−20°C), which allowed greater current to be tolerated during electrophoresis. PEGMAC gels enabled us to perform sixfold faster electrophoretic separations and achieve threefold improved resolution of six standard proteins at the lower temperatures in a direct comparison with the normal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) system. If this approach is coupled with more precise instrumentation to control low temperatures during electrophoresis, greater separation speeds and resolution may be anticipated.
Separation of long DNA fragments by capillary gel electrophoresis with laser‐induced fluorescence detection
Authors: Nishikawa Tetsuo , Kambara Hideki
Published: May 2005

Long DNA fragments were separated by capillary gel electrophoresis with laser‐induced fluorescence detection, and two approaches to obtaining high‐resolution separation of DNA fragments were examined. One method achieved high resolution at the expense of detection time, while the other achieved both high resolution and fast detection. The former approach used a 300 cm long capillary with a gel concentration of 4% T and an electric field strength of 70 V/cm. The resolution limit in this electrophoresis was 800 bases with a resolution value of 0.5 for adjacent peaks. Under these conditions, the measurement time was 44 h. The latter approach used a 200 cm long capillary with a gel concentration of 3% T and an electric field strength of 170 V/cm. The resolution limit in this electrophoresis was 680 bases with a resolution value of 0.5 for adjacent peaks, and the measurement time was only 10 h. With both approaches, fragments with less than 600 bases were efficiently separated. The resolution values for adjacent peaks with less than 500 bases are greater than 1.0, and those for peaks with less than 100 bases are greater than 2.7. These approaches thus improve the accuracy of the base sequence determination.
Effect of operational variables on the separation of proteins by capillary sodium dodecyl sulfate‐gel electrophoresis
Authors: Guttman András , Shieh Paul , Hoang Dao , Horváth Judith , Cooke N.
Published: May 2005

Capillary sodium dodecyl sulfate (SDS)‐gel electrophoresis, for fast and high‐resolution separations of protein molecules based on their molecular masses was investigated, employing a polymeric sieving network of polyethylene oxide (PEO). A standard mixture of five proteins ranging from 14.2‐66.0 kDa in molecular mass was used to evaluate the sieving matrix. A general migration velocity equation was derived for capillary SDS‐gel electrophoresis of proteins and supported by the experimental data. This equation gives a better understanding of SDS‐protein separations via capillary gel electrophoresis. Results are presented regarding the effects of different operational variables such as gel concentration, electric field strength, molecular mass, and temperature on the electrophoretic migration properties of the different size protein molecules.
Capillary electrophoresis monitoring of the competitive adsorption of albumin onto the orosomucoid‐coated polyisobutylcyanoacrylate nanoparticles
Authors: Olivier Jean‐Christophe , Taverna Myriam , Vauthier Christine , Couvreur Patrick , Baylocq‐Ferrier Danielle
Published: May 2005

The simultaneous and rapid quantitation of a glycoprotein, human orosomucoid, and a protein, human serum albumin (HSA), by micellar electrokinetic capillary electrophoresis was developed and optimized (pH and sodium dodecyl sulfate concentration) in order to study the competitive adsorption of HSA onto orosomucoid‐coated nanoparticles of polyisobutylcyanoacrylate. This method provided satisfactory repeatability and the response was linear for concentrations of orosomucoid and HSA ranging from 0.025 to 0.500 mg/mL (r > 0.994 for both proteins). To study the competitive adsorption of HSA, the amounts of free orosomucoid and free HSA were determined in the supernatant obtained after centrifugation of the orosomucoid‐coated nanoparticle suspension incubated with HSA. Furthermore, the quantities of both orosomucoid and HSA adsorbed onto nanoparticles were also determined after desorption of these molecules from the nanoparticle surface, using sodium dodecyl sulfate. No analytical interference was observed with this surfactant. The results showed that HSA did not at all adsorb onto the orosomucoidcoated nanoparticles and, thus, that this protein did not displace the layer of adsorbed orosomucoid from the nanoparticle surface.
Analysis of the proteins of aging Caenorhabditis elegans by high resolution two‐dimensional gel electrophoresis
Authors: Vanfleteren Jacques R. , De Vreese Annemie
Published: May 2005

The metabolic rate decreases dramatically as a function of age in the nematode worm Caenorhabditis elegans. Superoxide anion production, which is tightly linked to oxygen consumption, and thus to metabolic rate, drops to a 20‐fold lower level in 10‐day‐old, senescent worms, as compared to 4‐day‐old young adults. In a long‐lived mutant strain of the same species metabolic activity is much better preserved. High resolution two‐dimensional gel electrophoresis was employed to study alterations in the protein profile, correlating with changes of metabolic activity. Surprisingly, few proteins show age‐ or age‐ and strain‐specific variations of spot intensity. The abundance of the huge majority of proteins displayed on these gels remains unaltered, irrespective of age and strain differences. These results imply that there are no major age‐related alterations of proteins due to faulty protein synthesis or free radical attack, and that age‐related changes in the rate of protein synthesis and breakdown must be strictly coordinated throughout the aging process.
Two‐dimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: Application of mass spectrometry to peptide‐mass fingerprinting
Authors: Ji Hong , Whitehead Robert H. , Reid Gavin E. , Moritz Robert L. , Ward Larry D. , Simpson Richard J.
Published: May 2005

Protein patterns of normal human colonic crypts, isolated from different regions of the large intestine, and several colorectal cancer cell lines were compared using two‐dimensional electrophoresis gels (2‐DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue staining, the protein patterns for normal crypts isolated from the ascending, and descending, regions of the colon and the rectum, were almost (> 95%) identical. While 75–80% of the protein spots from normal crypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt‐like structures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetectable in the protein pattern from normal cells), proteins a (Mr ∼ 18 000, pI 6.7–6.9) and b (Mr ∼ 24 000, pI 5.9–6.0), were detected in the 2‐DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be considered as potential colon tumor markers. Approximately 60% of the cellular proteins from LIM 1215 cells, a colon carcinoma cell line that exhibits many properties associated with columnar cells, can be matched with LIM 1863 cells. The results presented here represent an initial phase in our efforts to develop a comprehensive protein database for normal human colon cells and several colorectal cancer cell lines. While our initial protein identification relied on microsequencing methodologies, we are presently evaluating peptide‐mass fingerprinting, utilizing capillary reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and electrospray mass spectrometry, as a means for rapid identification of proteins at subpicomole levels. Using this approach, protein #3 (Mr ∼ 66 000, pI 6.2) was identified as heat shock protein 60 from as few as seven tryptic peptide masses when they were screened against the molecular weight search (MOWSE) peptide‐mass database.
Identification of transformation sensitive proteins recorded in human two‐dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing
Authors: Rasmussen Hanne H. , Mørtz Ejvind , Mann Matthias , Roepstorff Peter , Celis Julio E.
Published: May 2005

A comprehensive human keratinocyte two‐dimensional (2‐D) gel protein database has been established to study the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease, cancer included. A major task in establishing such a database is to identify known proteins in the 2‐D gel patterns as well as to reveal hitherto unknown proteins. To date, protein identification has been performed by one or a combination of the following methods: (i) comigration with known proteins, (ii) Western blotting using specific antibodies, (iii) microsequencing and (iv) vaccinia virus expression of full length cDNAs. Recently, the systematic identification of proteins has gained a new dimension with the advent of computer programs for searching peptide molecular mass databases with experimentally obtained peptide mass maps. Here we investigate this approach to identify proteins that are highly up‐ or down‐regulated in simian virus SV40 transformed human keratinocytes (K14). Peptide mass maps of several proteins, including keratins 7, 8, 18 and 19 were obtained either by plasma desorption mass spectrometry (PDMS) analysis of high performance liquid chromatography (HPLC) purified peptides or by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) of total digests. The results demonstrated that peptide mass maps can be used for a rapid and sensitive protein identification allowing fast screening of proteins recorded in 2‐D gel databases. The mass spectrometric approach when combined with microse‐quencing strengthened identification, and added the possibility of full characterization of post‐translational modifications and sequence variations.
Purification and identification of tyrosine‐phosphorylated proteins from B lymphocytes stimulated through the antigen receptor
Authors: Gold Michael R. , Yungwirth Tom , Sutherland Claire L. , Ingham Robert J. , Vianzon Daisy , Chiu Readman , Van Oostveen Inge , Morrison Hamish D. , Aebersold Ruedi
Published: May 2005

The activation of protein tyrosine kinases (PTKs) and subsequent tyrosine phosphorylation of cellular proteins is a critical initial signal in the response of eukaryotic cells to mitogens, differentiative signals, and other stimuli. A number of PTK substrates have been identified and many of these are components of signal transduction pathways that regulate cell function. However, the majority of proteins that are tyrosine‐phosphorylated in response to receptor signaling remain unidentified. As some of these unidentified PTK substrates may also be signal‐transducing proteins, their identification and functional characterization is an important objective towards understanding receptor signaling. We describe the development of a comprehensive and general process for the isolation and structural characterization of tyrosine‐phosphorylated proteins. The method involves enrichment by anti‐phosphotyrosine affinity chromatography, electrophoretic concentration and separation, and proteolytic fragmentation of individual purified phosphoproteins. Resulting peptide fragments are separated by microbore reverse‐phase high performance liquid chromatography (RP‐HPLC) and a portion of the eluted peptides are subjected to electrospray‐mass spectrometry (ES/MS) for accurate determination of peptide masses. Proteolytic fragmentation of a protein produces a characteristic set of peptide masses that can be used to rapidly identify the protein by searching databases containing the peptide mass “fingerprints” for all known proteins. The identity of the protein established by this method can be confirmed by sequence analysis of selected peptides. We have applied this procedure to the analysis of PTK substrates from B lymphocytes that have been stimulated through the B cell antigen receptor (BCR). Signaling by this receptor is involved in the generation of antibodies against foreign molecules (antigens). The BCR activates multiple PTKs which phosphorylate at least 30 different proteins. We have identified several of these tyrosine‐phosphorylated proteins, including Syk, a PTK that is known to be tyrosine‐phosphorylated in activated B cells. Thus, the procedure described here can be used to identify regulatory proteins of low abundance. The process consists of a logical succession of compatible steps that avoids pitfalls inherent to prior attempts to characterize low abundance phosphoproteins and should find wide use for the identification of tyrosine‐phosphorylated proteins in other cell types.