bluuebunny/crossref_metadata_2025 huggingface dataset:
The last but not the least: The origin and significance of trailing adhesions in fibroblastic cells
Authors: Rid Raphaela , Schiefermeier Natalia , Grigoriev Ilya , Small J. Victor , Kaverina Irina
Published: May 2005

Mature adhesions in a motile fibroblast can be classified as stationary “towing” adhesions in the front and sliding trailing adhesions that resist the traction force. Adhesions formed at the front of motile fibroblasts rarely reach the trailing zone, due to disassembly promoted by intensive microtubule targeting. Here, we show that the majority of adhesions found at the trailing edge originate within small short‐lived protrusions that extend laterally and backwards from the cell edge. These adhesions enlarge by sliding and by fusion with neighboring adhesions. A further subset of trailing adhesions is initiated at a novel site proximal to trailing stress fibre termini. Following tail retraction, trailing adhesions are actively regenerated and the stress fibre system is remodeled accordingly; the tensile forces elaborated by the contractile actin system are consequently redirected according to trailing adhesion location. We conclude that persistent and dynamic anchorage of the cell rear is needed for the maintenance of continuous unidirectional movement of fibroblasts. Cell Motil. Cytoskeleton 61:161–171, 2005. © 2005 Wiley‐Liss, Inc.
Bystander cell proliferation is modulated by the number of adjacent cells that were exposed to ionizing radiation
Authors: Gerashchenko Bogdan I. , Howell Roger W.
Published: May 2005

Background

Direct cell‐to‐cell contact appears to be a prerequisite for the proliferative response of bystander WB‐F344 cells co‐cultured with irradiated cells; however, neither gap junctional intercellular communication nor long‐range factors released into the medium appear to be involved (Cytometry 2003;56A:71–80). The present work investigated whether the proliferative bystander response depends on the number of irradiated cells (cells exposed to external γ‐rays or cells exposed to short‐range β‐particles emitted by DNA‐incorporated 3H‐thymidine) that are adjacent to unirradiated bystander cells.

Methods

Subconfluent monolayers of rat liver epithelial cells (WB‐F344) were incubated in the presence of (methyl‐3H)thymidine at a concentration of 5.8 kBq/ml for 18 h. Radiolabeled cells containing 0.7 × 10−3 Bq/cell (absorbed dose: 0.14 Gy) were plated together with unlabeled cells in proportions of 6% and 94%, 12% and 88%, 25% and 75%, 50% and 50%, and 75% and 25%, respectively, keeping constant the total number of plated cells. In a parallel experiment, cells acutely exposed to 5 Gy of 137Cs γ‐rays were plated with unirradiated cells in the same proportions. In both experiments, cells were co‐cultured for 24 h followed by a flow cytometric study of their proliferation. The two cell populations in the co‐cultures were distinguished by staining one population with carboxyfluorescein diacetate, succinimidyl ester, which metabolizes intracellularly.

Results

Increasing the fraction of irradiated cells relative to unirradiated bystander cells led to an increase in proliferation of bystander cells. Specifically, in co‐cultures in which irradiated cells were initially mixed with unirradiated cells in proportions of 50% and 50% and of 75% and 25%, respectively, bystander cells showed a statistically significant increase of their proliferation compared with the controls.

Conclusions

The proliferative response of WB‐F344 bystander cells is modulated by the number of adjacent cells that are exposed to ionizing radiation from external γ‐rays or intracellularly emitted 3H β‐particles. © 2005 Wiley‐Liss, Inc.
Protein adsorption at glassy carbon electrodes: The effect of covalently bound surface groups
Authors: Downard Alison J. , Roddick Alisa D.
Published: May 2005

A diazonium salt coupling procedure was used to covalently bind various p‐substituted phenyls to glassy carbon electrodes. The response of modified electrodes for oxidation of ferrocenemethanol in the absence and presence of bovine serum albumin was monitored to give a measure of protein adsorption. Carboxylate groups close to the surface did not influence protein adsorption (relative to polished glassy carbon electrodes), however, hydrophobic groups increased adsorption. These results are compared to those for similar experiments at electrochemically pretreated glassy carbon electrodes. Hydrophilic groups attached to the surface via sufficiently long linkages reduced protein adsorption and may be useful for analysis of clinical samples.
Interpreting diffusion coefficients of electrochemical probes in oil‐in‐water microemulsions
Authors: Mackay Raymond A. , Myers Stephanie A. , Brajter‐Toth Anna
Published: May 2005

The relationship between apparent diffusion coefficients, D′, of electroactive probe and thc microstructure of an oil‐in‐water anionic microemulsion, sodium dodecyl sulfate (SDS)/dodecane/I‐pentanol/0.1M NaCIaq has been investigated. Three probes, ferrocene (Fc), Fe(CN) and methyl viologen (MV2+) allowed the effect of ditrerent microstructure and interactions on the D′‐values to be investigated. The results indicate that the droplet as well as hicontinuous microemulsion structures can be identified and point to the effects of cross‐ phase exchange, partitioning, and multiple binding on the D′‐values.
Horizontal two‐dimensional (iso‐dalt) electrophoresis: Use of agarose isoelectric focusing and SDS gel electrophoresis in an exponential gradient for characterization of plasma proteins
Authors: Emerson David L. , Chapuis‐Cellier Colette , Arnaud Philippe
Published: May 2005

The two‐dimensional (iso‐dalt) electrophoresis system for protein separation has been modified to include the use of flat‐bed agarose isoelectric focusing in the first dimension, horizontal SDS‐electrophoresis in an exponential acrylamide gradient in the second dimension, molecular weight standards run side by side with the sample in the SDS‐polyacrylamide gel, and radiolabeled proteins as internal standards. Direct determination of the pH gradient after the first dimension and of molecular weights after the second dimension permits more accurate measurement of the protein spots in both the x and y axes, thus improving the confidence limits of the technique. The reproducibility of molecular weight determinations was calculated for eight different proteins in six different gels, with an overall standard deviation of 3.02%. Incorporation of internal standards is useful in identifying and confirming protein patterns. Examples of the application of this technique to the separation of specific plasma proteins are described.
A comparison of commonly used discontinuous and continuous buffer systems for electrophoresis in sodium dodecyl sulfate‐containing polyacrylamide gels
Authors: Lanzillo Joseph J. , Stevens Joanne , Fanburg Barry L.
Published: May 2005

A comparison between commonly used multiphasic zone electrophoresis (MZE) and continuous zone electrophoresis (CZE) buffer systems for polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS‐PAGE) was performed. Some proteins behave differently in one or more of the buffer systems, indicating that buffer choice for SDS‐PAGE may be more important than previously recognized. Guidelines are suggested for choosing buffer systems for purposes other than molecular weight analysis.
Continuous flow electrophoresis: The crescent phenomena revisited Part II: Nonisothermal effects
Authors: Ivory Cornelius F.
Published: May 2005

An asymptotic solution to the partial differential equation governing solute behavior in the continuous flow electrophoresis device is combined with solutions for the convective, electrophoretic and electroosmotic velocities in the chamber under nonisothermal operating conditions. The results illustrate the effect of temperature gradients on the net dispersion of solute in continuous flow electrophoresis. Three modes of operation are tested for this chamber: the convective velocity is in the same direction as gravity, the convective velocity is in the direction opposite to gravity and the effect of gravity is negligible.
A simple procedure for high efficiency counting of labeled protein bands in polyacrylamide slab gels
Authors: Chao Lee , Chao Julie
Published: May 2005

A simple procedure is described for counting labeled protein bands after polyacrylamide slab gel electsrophoresis. Proteins labeled with radioisotopes are separated on conventional polyacrylamide slab gels. After fiction and staining, the slab gels are dried onto filter papers under vacuum. The labeled protein patterns are recorded by autoradiography or fluorography. The sample tracks are separated from each other by cutting the dried slab gel lengthwise. Each sample track is cut into predetermined sections by making parallel cuts with a film cutter. The sections are deposited into scintillation vials and digested with 50% hydrogen peroxide. The digested samples are counted in an aqueous scintillator. This procedure provides a direct quantitation of 3h and 14C‐labeled protein bands by reproducibility counting. The results also show that both high resolution and excellent reproducibility are obtainable by this relatively fast and inexpensive method.
Rapid and sensitive location of protein zones in gels using o‐phthaldialdehyde
Authors: Taylor Michael D. , Andrews Anthony T.
Published: May 2005

A new fluorescent staining technique using o‐phthaldialdehyde(OPA) has been developed for the rapid post‐electrophoretic location for separated zones of proteins and glycoproteins. The method has also been applied to the detection of immunoprecipitates and, in the form of a spray reagent, to the detection of proteins separated on thin‐layer gel filtration plates. Major protein zones can be visualized within 1–2 min with maximum sensitivity being achieved within 15 min. In polyacrylamide and agarose gels, the sensitivity is of the same order as staining with Coomassie Blue G‐250. Reagents are very inexpensive and, because the reaction is performed at neutral or weekly alkaline pH in aqueous buffers, retention of enzymatic or other biological activity should be optimal. The method is thus particularly suitable for use in preparative applications and when it is desirable to combine general protein detection with specific location of zone of enzyme activity.
Electrophoresis in gels containing zwitterionic groups
Authors: Hjelmeland Leonard M. , Allenmark Stig , An der Lan Birgit , Jackiw B. Andrew , Nguyen Nga Y. , Chrambach Andreaas
Published: May 2005

A zwitterionic methacrylamide derivative, (3‐sulfopropyl)dimethy1(3‐methacrylamidopropyl) ammonium inner salt (MAPS), was synthesized and then copolymerized with N,N′‐methylenebisacrylamide (Bis) to make gels carrying covalently bound sulfobetaine groups. Gels were also prepared with the addition of (3‐sulfopropyl)trimethyl ammonium inner salt (TMAPS) to Bis‐crosslinked polyacrylamide prior to polymerization. The properties of these gels in gel electrophoresis and electrofocusing were tested and compared with those of Bis‐crosslinked polyacrylamide.

In MAPS gels the absolute migration velocities of proteins and dyes at either polarity of migration were decreased. Anionic species were much more strongly retarded than cationic species. Ferguson plots (based on protein mobilities relative to dye in a continuous buffer) of catalase at pH 4 obtained on MAPS gels had a similar slope but lower y‐intercept compared with those on equivalent polyacrylamide gels, indicating that the available (effective) net charge on the protein was decreased because it interacts with the zwitterionic charge on the gel. Similarly, the addition of TMAPS to polyacrylamide decreased the relative catalase mobility at non‐restrictive gel concentrations. However, at gel concentrations above 4 %T, relative mobilities were increased, presumably because the effective pore size of the gel was increased through inhibition of polymerization by the zwitterionic compound.

MAPS gels did not exhibit electroendosmosis by the criterion of cyanocobalamin displacement. Resolution between peptides in MAPS gels, and between proteins in ternary copolymer gels made of MAPS, Bis and polyacrylamide, was similar to that on polyacrylamide. Bands of catalase appeared sharpened in MAPS gels. pH Gradients form, decay and exhibit conductance gaps in MAPS electrofocusing gels and in polyacrylamide gels in presence of 0.25‐1.5 M TMAPS, in a manner which was qualitatively indistinguishable from polyacrylamide.
Electrophoresis of viral proteins in formic acid 1. General aspects
Authors: Heukeshoven Jochen , Dernick Rudolf
Published: May 2005

The excellent solublizing properties of high concentrations of formic acid solutions for polypeptides and proteins, including those with high hydrophobicity, are used in polyacrylamide gel electrophoresis. The technical handling of the system is described in detail. The structural polypeptide of poliovirus type1, VP1, VP2, VP3 an dVP4, until now separable in polyacrylamide gel rods containing 70% formic acid. By two‐dimensional analysis, namely formic acid electrophoresis in the first dimension and SDS electrophoresis in the second, we have shown that the electrophoretic mobility in formic acid is of the order VP2‐VP1‐VP3‐VP4. with several marker proteins we have also demonstrated that the migration velocity depends not only on the molecular weights but also on the secondary structure of the proteins, especially on disulfide cross‐links. High concentrations of salts (3 MCsCI or 6 m Guanidine hydrochloride) in the protein sample do not change the separation pattern, which, however, is strongly affected by 6 m urea.
Peptide maps of labeled poliovirus proteins after two‐dimensional analysis by limited proteolysis and electrophoresis in sodium dodecyl sulfate
Authors: Wiegers Klaus J. , Dernick Rudolf
Published: May 2005

A simple technique for characterization and identification of proteins after high resolution two‐dimensional (2D) electrophoresis is described. Poliovirus proteins were separated by 2D analysis and then subjected to llimited proteolysis in sodium dodecyl sulfate (SDS) by alfa‐Chymotrypsin or Staphylococcus V8 protease. The cleavage products were analyzed by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). The pattern of peptides obtained was characteristic for a given protein and for the protease used and was highly reproducible. Alfa‐Chymotrypsin produced fewer and larger peptides and was suitable for large poliovirus proteins. Staphylococcus V8 protease yielded smaller peptides and therefore showed a higher degree of discrimination between the poliovirus proteins analyzed. A precursor product relationship for a number of poliovirus proteins was determined by this simple technique.
Anamalous electrophoresis behavior of rebonuclease from human urine and a novel treatment method for reliable analysis
Authors: Thomas John M. , Hodes Marion E.
Published: May 2005

Since Desialylated rebonuclease (RNase) from human urine has a highle alkaline isoelectric point (pI 9.5–10.5), an acidic buffer system is required for electrophoretic analysis of different urine specimens. In a common acid disc electrophoresis system in thin‐layer polyacrylamide gel, the cathodal mobility of the RNase enzyme(s) was markedly reduced from that expected on the basis of the estimated pI. Dialysis urine against water had a very detrimental effect on the separation of the isozymes, and the addition of buffer to the dialyzed urine further exacerbated this effect. The urin RNase exhibited anomalous electrophoretic behavior in the form different urine specimens or from the same urine specimen adjusted to different concentrations. Treatment of urine with various cations had a significant, positive effect on the migration and resolution of the isozymes. This and other experimental evidence is consistent with the hypothesis that the anomalous behavior was due to the binding of anions from the buffer. A standardized protocol was developed for processing urine specimens, including treatment of urine with myoglobin, which abolished the artifactual variation observed previously and enabled reliable electrophoretic separation of the urine RNase isozymes. In addion, myoglobin treatment had a similar positive effect on the electrophoretic behavior of RNase enzymes from some other sources.
Analysis of bacteriophage T4 coat proteins by two‐dimensional electrophoresis and computerized densitometry
Authors: Gersten Dogulas M. , Kurian Ponnamma , Ledly Gary , Park Chan M. , Suhocki Paul V.
Published: May 2005

We have designed treatment conditions which allow analysis of the coat proteins of bacteriophage T4D+ by high resolution two‐dimensional electrophoresis. The best results were obtained when the mature phage particles were subjected to hypertoniclysis and DNase treatment before solubilization with sodium dodecyl sulfate (SDS). Samples containing 250 ug protein gave the optimum resolution, producing 45 spots as visualized by Coomassie Blue staining. Computerized densitometry measurements showed that these proteins range in relative abundance from less than 0.22% to 11.8% of the total.
The fabrication of multichannel tubing and its use in a continuous‐flow isielectric focusing apparatus to give smooth laminar flow
Authors: Quarmby Christopher
Published: May 2005

Details are given of a method of casting multichannel tubing in silicone rubber in the form of a slab 2 mm thick and up to 380 mm long. Up to 264 parallel channels were formed at 0.8 mm centers along the length of the material, each channel being 0.4 mm in diameter. The material was used both as the outlet manifold in an apparatus for continuous‐flow isoelectric focusing (IEF), the construction of which is described, and also in conjunction with a slow‐running peristaltic pump as multichannel pump‐tubing when it was found that at a flow‐rate of about 0.3 ml h−1 per channel, the variation between channels was less than 0.6%. The coupling of the pump‐tubing to the outlet manifold of the separation cell of the IEF apparatus resulted in a very smooth laminar flow of liquid in the separation cell itself.
Isolectric patterns of human alpha1‐ antichymotrypsin (A1AChy) and A1AChy‐protease complexes
Authors: Gianazza Elisabetta , Arnaud Philippe
Published: May 2005

When studied by thin‐layer polyacrylamide gel isoelectric focusing, alpha1‐an‐tichymotrypsin (A1AChy) in human plasma presents a microheterogeneous pattern, consisting of seven bands with isoelectric points (pI) between 4.1 and 4.45. After removal of sialic acids by neuraminidase treatment, four bands are seen with a more alkaline pI (about 1.0 pH unit). Thus, the microheterogeneity of human A1AChy is due only in part to differential sialylation of the isoproteins. Incubation of A1AChy (in excess) with alpha‐chymotrypsin results in the formation of a primary complex. In the presence of excess protease, incubation results in a secondary complex with a lower pI. Upon incubation of A1AChy with trypsin, no protease‐inhibitor complexes are observed, although there is evidence for partial degradation of the A1AChy molecule.
Electrophoresis of adsorbed albumin
Authors: Absolom Darryl R. , Michaeli Isaac , van Oss Carel J.
Published: May 2005

It was recently observed that bovine serum albumin (BSA), adsorbed onto part of the surface of a glass slide, diffuses along the unoccupied part of that surface (without redissolving in the bulk liquid) at a much greater velocity than the normal diffusion rate of BSA, free solution. In this work a study is made of the electrophoretic transphoretic transport of BSA, adsorbed onto part of the surface of glass slide, along the hitherto unoccupied part of the glass surface. The electrophoretic mobility of adsorbed BSA along the unoccupied glass surface proved to be up to ≅ 2 times faster than in electrophoresis on cellulose acetate strips at the same pH and ionic strength, and up to ≅ 1.6 times faster than in moving boundary electorphoresis. The increase in electrophoretic mobility of adsorbed BSA is most pronounced at the highest density of adsorption. At that density the oblong BSA molecules are closely packed, most probably adsorbed in a tight monolayer, with each molecule positioned perpendicular to the glass surface (although other configurations cannot be excluded). The major driving force enhancing the diffusivity, as well as the electrophoretic mobility of adsorbed BSA, appears to be the high surface pressure of he tightly packed BSA layer. A tentative explanation is offered for the low resistance BSA molecules encounter in moving parallel to the glass surface while remaining adsorbed to the liquid, involving free energies of adhesion of individual protein molecules in various orientations.
Determination of molecular weights and Stokes' radii of non‐denatured proteins by polyacrylamide gradient gel electrophoresis. 2. Determination of the size of stable and labile molecular weight variants of enzymes from plant sources
Authors: Rothe Gunter M. , Purkhanbaba Huschang
Published: May 2005

Under certain conditions in polyacrylamide gradient gel electrophoresis (PAGGE), a linear correlation between the logarithm of the size of calibration proteins (log MW or log Rs) and the square root of their migration distance (√D) can be observed; slope and intercept of the calibration curve depend on the duration of electrophoresis; linearity, however, is maintained over a wide range (4‐60 h, 200 V) (Rothe and Purkhanbaba, Electrophoresis 1982, 3, 33–42.) Using this method the reaction of plant isozyme systems penetrating a linear polyacrylamide (PAA) gradient gel was investigated: lactate dehydrogenase (LDH) from potato tubers behaves similarly to animal calibration proteins. The enzyme exhibits 3 subbands with molecular weights of 144 000, 150 000 and 156 000 (± 1.7 %), respectively. The enzyme system “shikimate oxidoreductase” (SORase) behaves significantly different: during PAGGE it splits into 2 major bands (∼60 000 and ∼110 000, respectively) consisting of several subbands. The results of our experiments show that SORase is present in vivo as a complex system. We believe that this complex is partly indentical with the so‐called pre‐chorismate complex.
Electroimmunochemical analysis of amphiphilic proteins and glycolipids stained with Sudan Black‐containing detergent micelles
Authors: Bjerrum Ole J. , Gerlach James H. , Bøg‐Hansen Thorkild C. , Hertz Jesper B.
Published: May 2005

Micelles of non‐ionic detergents can be colored through incorporation of the hydrophobic dye Sudan Black. This property can be used to identify amphiphilic antigens in complex mixtures. Thus, crossed immunoelectrophoresis with 0.05 % v/v Triton X‐100 plus 0.002 % w/v Sudan Black shows selective staining of major amphiphilic proteins from human erythrocytes, thrombocytes and serum, from bovine milk fat globules and from yeast plasma membranes. The method may also give information about glycolipid antigens in detergent‐solubilized membrane material, e.g. mannosyl‐diinositolphosphoryl‐ceramide from yeast. In this case, however, the membrane has to be solubilized with detergent plus Sudan Black and analyzed in detergent‐free gels, where a stable micellar structure seems to exist. In a surplus of detergent, the glycolipid antigen may enter new micelles during electrophoresis, reducing the number of epitopes per micelle below two, thus avoiding precipitation. Analysis of purified lipopolysaccharide from Bordetella pertussis followed this pattern, whereas lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli, although heavily influenced by the presence of Triton X‐100, were precipitated in the presence of a surplus of detergent. The described method is simple, fast and easy to perform. It requires neither extra equipment nor expensive chemicals. Sometimes heavily stained, detergent‐containing precipitates of a non immunological nature may appear, but these can easily be discerned from immunoprecipitates.
Steady‐state stacking in agarose at various pH
Authors: Buzás Zsuzsanna , Chrambach Andreas
Published: May 2005

The applicability of multiphasic buffer systems to electroendosmosis‐free agarose (IsoGel) was tested in order to be able to combine the benefits of agarose gel electrophoresis with those of steady‐state stacking. It was found that buffer systems of a negative polarity function normally over the entire pH range, whereas proteins with positive net charge are retarded at the gel surface in buffer systems with positive polarity. This retardation is not due to a failure to establish a moving boundary in agarose. Rather, it appears due to a negatively charged weakly acidic functional group in agarose which can be removed from agarose by pre‐electrophoresis at 0.1 M ionic strength. After its removal positively charged proteins also stack normally in multiphasic buffer systems of the positive polarity.
Isoelectric focusing pattern of human antithrombin III (AT‐III): Effects of heparin or thrombin on a microheterogeneous form of AT‐III
Authors: Kera Yoshio , Yamasawa Kichihei
Published: May 2005

After agarose gel isoelectric focusing (AGIEF) and immunofixation, human antithrombin III (AT‐III) in plasma showed a microheterogeneous pattern which consisted of 5 major and 5 minor components with isoelectric points (pI) between pH 4.8 and 5.2. Treatment with neuraminidase reduced this microheterogeneity of AT‐III to 1 major, 3 sub‐major and 2 or 3 minor components with a shift in pI to between pH 5.5 and 5.8. Thus, differential sialylation of the isoproteins partially contributed to the microheterogeneity observed. The addition of heparin to plasma produced broad acidic precipitates with pIs between pH 4.2 and 4.7 on the gel. The same acidic precipitates were also obtained from heparin‐purified AT‐III mixture. The appearance of these precipitates was related to the decreasing in number or in staining intensity of the native AT‐III bands. These precipitates may be complexes of heparin and AT‐III. The addition of human or bovine thrombin to plasma produced several basic minor components, which were similar to those in serum, at a thrombin concentration of 48 or more NIH U/ml‐plasma. The basic minor components were not obtained from a purified AT‐III‐thrombin mixture. These might be complexes between AT‐III, thrombin and some of the other coagulation factors.
Comparative analysis of subcellular proteins by SDS‐polyacrylamide gel electrophoresis with dorsolateral and ventral prostates of rats
Authors: Matuo Yuhsi , Nishi Nozomu , Negi Tetsuro , Wada Fumio
Published: May 2005

Proteins in the cytosol, post‐nuclear particulate and nuclear fractions from dorsolateral and ventral prostates of rats were analyzed and compared by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. Protein species separated in the gel were stained with Coomassie Brilliant Blue R‐250 (CBB), and expressed as (apparent molecular weight of the respective species x 10−3)K. (i) In the cytosol fraction the contents of 30K, 67K, 110K, and 120K proteins were abundant in the dorsolateral prostate. On the other hand, 14K and 16K proteins were specific to the ventral prostate. By carbohydrate staining, major glycoproteins were found to be 120K and 16K for the dorsolateral and ventral prostates, respectively. (ii) In post‐nuclear particulate fractions in both lobes, proteins having molecular weights greater than 42 000 were abundant. Of these, the contens of 65K, 105K and 120K were higher in the dorsolateral prostate than in the ventral prostate. Glycoproteins having the same apparent molecular weight as detected in the cytosol fractions were also found in the post‐nuclear particulate fractions of both lobes. (iii) In the nuclear fraction H2A, H2B, H3 and H4 histones were fundamentally similar between the two lobes, whereas H1 histones including two subspecies were slightly but significantly higher in the ventral prostate than the dorsolateral prostate. The most remarkable difference between the lobes was the content of 20K in the dorsolateral prostate which was at least 20 times higher than that found in the ventral prostate.
Isolation of urinary β2‐microglobulin by isoelectric focusing: Use of a new method of protein localization
Authors: Bonitati James , Sabatino Bruce , Van Liew Judith B.
Published: May 2005

An indirect fluorescence method for locating colorless protein bands in focused Sephadex gel slabs, demonstrated previously using standard protein solutions [1, 2], has been applied to a complex natural protein mixture: preconcentrated human urine. Protein bands separated by distances of 2 mm or less in the gel slab were accurately located and excised without cross‐contamination. Urinary pigment did not interfere with the localization technic. Microgram quantities of six protein bands were excised from thin gel slabs, and then partially characterized by cellulose acetate electrophoresis. In addition, the bands belonging to two homologues of β2‐microglobulin (B2M I and II) were identified immunologically after systematic excision of the various protein bands in the focused pattern. This one‐step protein isolation procedure was scaled up to the milligram level for the isolation of B2M‐II. In two experiments, 1.60 mg and 2.22 mg were isolated in 73 % and 78 % purity, with yields of approximately 55 % and 76 %.
Analysis of the genetic variants of the human Gc system (VDBP) by isoelectric focusing in immobilized pH gradients
Authors: Cleve Hartwig , Patutschnick Walburgis , Postel Wilhelm , Weser Johann , Görg Angelika
Published: May 2005

The genetic variability of the human group‐specific component (Gc) system has been examined by isoelectric focusing with immobilized pH gradients. The phenotypes of the six common Gc types and of 15 rare genetic Gc variants are described and presented. High resolution of the Gc components is accomplished. The classification can be made without specific immune reaction. Isoelectric focusing with immobilized pH gradients represents a powerful tool for analyzing genetic variability.
Description and detection of moving sample zones in zone electrophoresis: Zone spreading due to the sample as a necessary discontinuous element
Authors: Thormann Wolfgang
Published: May 2005

This paper deals with the description and detection of moving sample zones in free zone electrophoresis. It is shown that the shape of a sample zone never reaches a steady‐state. However, either the front or the rear boundary of the sample zones, but not both, can represent steady‐state concentration distributions with decreasing peak heights. This results in an unsymmetrical broadening of the moving zones. With a multichannel detection device, consisting of 255 equidistant detectors along the separation trough, asymmetric electric field distributions of the sample components are measured. These are shown to agree with theoretical predictions, made by a model based on migration only. This clarifies the fact that in zone electrophoresis migrational dispersion due to the applied electric field is usually much larger than diffusional spreading.
Staining of Concanavalin A‐reactive glycoproteins on polyacrylamide gels with horseradish peroxidase — a critical evaluation
Authors: Schott Klaus‐Joachim , Neuhoff Volker , Nessel Birgit , Pötter Ulla , Schröter Joachim
Published: May 2005

The method to localize and quantitate glycoproteins on polyacrylamide gels is based on the binding of Concanavalin A to the glycoproteins and the subsequent coupling of horseradish peroxidase to the glycoprotein ‐ Concanavalin A complex. The complex is visualized by the oxidation of 3,3 ‐diaminobenzidine with horseradish perexidase ‐ H2O2, forming a brownish stain at the gel surface. Various parameters are analyzed and a scheme for the optimal staining of macro and micro slab gels is given. The quantitative evaluation of the stained gels is demonstrated. The staining and quantitation of nitrocellulose transfers is also shown.
A photodevelopment silver stain for the rapid visualization of proteins separated on polyacrylamide gels
Authors: Merril Carl R. , Harrington Michael , Alley Valerie
Published: May 2005

By using light (“photodevelopment”) to develop a metallic silver image of proteins separated on polyacrylamide gels, it is possible to visualize protein and nucleic acid patterns within 10 min after electrophoretic separation. This light catalyzed silver stain is based on establishing gel conditions in which ionic silver is reduced to silver metal in the presence of light energy at a different rate in gel regions containing biopolymers (such as proteins or nucleic acids) than the rate of reduction in regions which are devoid of such biopolymers. This photodevelopment method requires only two solutions: a solution to “fix” the proteins and a solution containing silver ions, which produces an image when exposed to light. This type of protein stain has achieved a sensitivity of 0.5 ng of protein. DNA separated on polyacrylamide may also be visualized with this stain. However, DNA generally produces a negative image. Studies of photodevelopment silver stains allow further insight into the processes which are involved in the ability of silver to form images of biopolymers separated by electrophoresis.
Bidirectional analytical isotachophoresis: Simultaneous determination of anionic and cationic boundaries
Authors: Thormann Wolfgang , Arn Dieter , Schumacher Ernst
Published: May 2005

This paper deals with electrolyte systems capable of producing suitable isotachophoretic zone structures in either migration direction. These systems contain moving reaction boundaries. A dilute solution of a strong acid as anolyte and the sodium salt of a weak acid as catholyte are analyzed to provide insight into the features of such dual systems. For various electrolyte arrangements the boundaries are examined for resolving capability in isotachophoresis. Both anionic and cationic boundaries were followed simultaneously using a capillary type apparatus with an array detector.
Long‐term storage of free and polyacrylamide gel‐bound Immobiline chemicals
Authors: Pietta Piergiorgio , Pocaterra Enrica , Fiorino Antonio , Gianazza Elisabetta , Righetti Pier Giorgio
Published: May 2005

The stability with time of Immobiline solutions and of pre‐cast Immobiline gels has been investigated, for the former by high pressure liquid chromatography and for the latter by focusing a set of marker proteins. Temperature seems to affect these chemicals most: whatever degradation occurs at 20°C within a few weeks' time, is operative at 60°C within a few hours' time. These buffers are altered by pH in different ways: acidic Immobilines (pKs 3.6 and 4.6) are extensively degraded around neutrality (pH 6) while basic species (especially pK's 8.5 and 9.3) are massively destroyed in alkaline solutions (pH 9). Acidic pH values (pH 3–4) ensure maximum stability for all Immobiline chemicals. In terms of medium‐term storage of pre‐cast gels, wet matrices exhibit least degradation if titrated to pH 4. Formic acid is the best titrant since it can be efficiently removed during the focusing step with formation of the narrowest salt‐front at the anode. Dry matrices have much longer stability (>2 months) but should be stored with traces of humidity to prevent the cracking and peeling‐off from the plastic support which occurs at zero relative humidity.
Quantitative measurements of fluorescently‐stained DNA in gels from photographic images
Authors: Willis Charles E. , Holmquist Gerald P.
Published: May 2005

Photography under ultraviolet illumination is used routinely to record the results of gel electrophoresis of DNA when a fluorescent dye, which binds to DNA, is present in the gel. The distribution of DNA in the gel can be determined accurately from the photographic negative when the absorbance is interpreted according to the nonlinear film darking function, when the contrast of the film is calculated, and when the images are corrected for uneven ultraviolet transillumination. The contrast can be calculated from the images of a fluorescent plastic intensity standard. The local intensity of the unbound‐dye fluorescence can be used to correct images for uneven illumination. Using this approach, relative abundances of DNA fragments, compared within each electrophoretic track, were measured within an average error of 6.6% of the expected value with no evidence of systematic error. Quantitative comparisons were valid anywhere on the film for a 50:1 range of intensities, with errors distributed normally with a standard deviation of ± 10 % of the expected value.
A comparison of two‐dimensional gel electrophoresis methods for analysis of rat serum proteins following dimethylformamide exposure
Authors: Marshall Thomas , Williams Katherine M. , Vesterberg Olof
Published: May 2005

Four two‐dimensional electrophoresis methods, each incorporating different protein denaturing/dissociating conditions, have been used to detect changes in rat serum proteins following exposure to dimethylformamide. The methods were evaluated on the basis of (i) their ability to detect changes, (ii) the resolution achieved and (iii) the response of resolution to the use of narrow Ampholine pH ranges in the first dimensional isoelectric focusing gels. All methods reveal minor but reproducible protein changes in response to dimethylformamide. However, high resolution two‐dimensional electrophoresis of the completely dissociated polypeptides reveals most changes and the resolution of this method is most improved by the use of Ampholines of narrow pH range. In general, the number of serum components detected and the resolution achieved is related to the severity of the protein denaturing/dissociating conditions employed. The significance of this observation in relation to others factors likely to influence the choice of two‐dimensional method is discussed.
Comparison of focusing in buffers and synthetic carrier ampholytes for use in the first dimension of two dimensional polyacrylamide gel electrophoresis
Authors: Burghes Arthur H. M. , Patel Ketan , Dunn Michael J.
Published: May 2005

Buffer focusing using the mixture Polybuffer 47 (Polysciences) has been tested for the separation of proteins under denaturing conditions to assess its potential for use as the first dimension in two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). a relatively simple pattern, with the proteins confined to certain regions of the gel, was observed when [35S]methionine‐labelled fibroblasts were separated both by 2D‐PAGE and by one‐dimensional isoelectric focusing (IEF) using Polybuffer 47. When Polybuffer 47 and synthetic carrier ampholytes were mixed, the latter were also found to concentrate into regions corresponding to those occupied by the proteins. The addition of synthetic carrier ampholytes resulted in separation of proteins within these regions. Diagonal IEF gels of synthetic carrier ampholyte versus buffer focusing showed a “step” pattern, suggesting that buffer focusing resulted in concentration of proteins in particular regions. Diagonal IEF gels using synthetic carrier ampholytes in both dimensions verified that protein‐ampholyte interactions are not a major source of artefacts in such gels. When mixtures of Polybuffer 47 and synthetic carrier ampholytes were used, there was some extension of the pH gradient at both ends. We conclude that Polybuffer 47 alone is not suitable for use in the first dimension of high‐resolution 2D‐PAGE separations.
Identification map of human plasma proteins: Micro two‐dimensional electrophoresis followed by multiple immunoreplica technique
Authors: Manabe Takashi , Takahashi Yuko , Higuchi Noriko , Okuyama Tsuneo
Published: May 2005

Human plasma proteins were subjected to parallel micro two‐dimensional polyacrylamide gel electrophoresis in the absence of denaturing agents. Forty nitrocellulose blots were obtained from eight slab gels within one hour of electroblotting time, employing an apparatus designed to obtain multiple replicas from micro slab gels. The locations of 31 human plasma proteins were determined on these blots using specific antisera, and an “identification map” was prepared. Differences of protein location between plasma and serum were also demonstrated.
The engineering design of continuous electrophoresis
Authors: Andrews Graham F. , Fonta Jean‐Pierre
Published: May 2005

In the design of continuous electrophoresis devices, the main variables are the gap width, the flow rate and inlet temperature of the buffer, the electric field strength, the point of injection of the proteins, and the orientation of the flow to the gravitational field. Best values for many of these can be derived from first principles by requiring that the flow be stable, and that streak spreading be minimized. The result is a downflow device, with larger gap width than is usual, warm inlet buffer solution, and protein injection slightly downstream of the start of the electrodes. If cooling is improved by increasing the thermal conductivity of the cooling walls, the gap width must be reduced and the buffer flow increased. The performance possible with perfect cooling walls is similar to that obtained in space because, in both cases, the limiting factor is not thermal convection but the high temperature generated at the mid‐point between the cooling walls.
An isotachophoretic analysis of the interaction of bilirubin and biliverdin with bovine serum albumin
Authors: Oefner Peter , Csordas Adam , Bartsch Georg , Grunicke Hans
Published: May 2005

The binding sites of non‐defatted and fatty acid‐free bovine serum albumin for bilirubin and biliverdin were investigated by means of analytical capillary isotachophoresis. In addition, the effect of oleic acid on the binding of bilirubin was examined with the same analytical tool. Albumin‐bilirubin complexes with increasing molar ratios of bilirubin can be identified as a separately migrating zone adjacent to the zone of free bilirubin. At pH 9 there are 3–4 high‐affinity binding sites for bilirubin and, possibly, several weaker binding sites. Fatty acid‐free and non‐defatted albumin exhibit almost identical binding properties with regard to bilirubin. Under the same conditions non‐defatted albumin shows only one high‐affinity binding site for biliverdin. The analysis of fatty acid‐free albumin‐biliverdin complexes reveals additional weak binding sites, which indicates that the physiological loading of albumin with ligands interferes only with the low‐affinity binding of biliverdin. At sufficiently high concentrations, oleic acid even competes for the first high‐affinity binding site of bovine serum albumin for bilirubin. There is a linear relationship between the zone length and the molar concentrations of free ligands (bilirubin, biliverdin). The differential binding characteristics of bilirubin and biliverdin indicate that the reduction of biliverdin yields a molecule which has a higher affinity for albumin; this, however, results in an inferior water solubility at physiological pH values. Owing to high resolution and quantification, capillary isotachophoresis is a powerful tool for screening endogenous and exogenous compounds with regard to their effect on the bilirubin transport capacity of albumin.
Ultrathin‐layer isoelectric focusing of the photoaffinity labeled benzodiazepine receptor in calf brain: A new and simple method for the study of integral membrane proteins
Authors: Lentes Klaus Ulrich , Friedl Waltraut , Hebebrand Johannes , Propping Peter
Published: May 2005

A new method for analytical isoelectric focusing of the benzodiazepine receptor in calf brain is described. Brain membranes are photoaffinity labeled with a specific radioligand, solubilized in 9 M urea and separated on 150 μm ultrathin polyacrylamide gels containing 8 M urea. The method combines high resolution with speed and versatility. It may be generally applicable for isoelectric focusing of integral membrane proteins in genetic studies.
Sensitive alkaline phosphatase linked secondary antibody system for detection of group specific component after isoelectric focusing on 250 μm thick reusable immobilized pH‐gradients
Authors: Pflug Werner
Published: May 2005

A sensitive secondary antibody system was developed for detection of small amounts of group specific component (Gc). For the first antibody step after focusig, a comparison between immunofixation on cellulose acetate membranes and capillary blotting on nitrocellulose was made. The sensitivity of the first is much better than that of the latter. The method allowed the detection of about 150 picograms of Gc in 1:12 800 diluted serum samples. Even in six‐year‐old badly soluble bloodstains kept at room temperature, Gc could be phenotyped. The 250 μm thick immobilized pH‐gradient gels for focusing of Gc can be used repeatedly after washing and drying to the initial weight without destroying the pH gradient.
Urine analysis by two‐dimensional gel eletrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension
Authors: Gianazza Elisabetta , Astrua‐Testori Silvia , Righetti Pier Giorgio , Bianchi‐Bosisio Adriana
Published: May 2005

Urine analysis by two‐dimensional gel electrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension allows the loading of high sample volumes and/or of large amounts of salts. When applied to urinary protein analysis, it makes it possible to apply up to 1 mL of dialyzed, unconcentrated sample or up to 350 μL of unprocessed urine.
HERMeS: A second generation approach to the automatic analysis of two‐dimensional electrophoresis gels. Part III: Spot list matching
Authors: Vincens Pierre , Tarroux Philippe
Published: May 2005

One of the most critical steps in two‐dimensional gel analysis is the comparison of spot lists. In this paper, we describe our approach to the matching problem. We propose a method based on syntactic pattern recognition techniques, which differs from previously used methods and offers a major improvement to cope with discontinuous geometric distortions. The method is based on non‐metric considerations and describes the pattern observed on a given gel in a non‐geometric way. This approach leads to the definition of finite automatons which can be used to automatically find the presence of a given pattern on a gel. The program uses a technique of clause generation related to artificial intelligence techniques. A set of rules is applied to the definition of homologousspots, and probability coefficients are computed to compare and to chose between different pairing hypotheses. Once matching has been achieved between a set of gels, the construction of a master gel which summarizes the information obtained is allowed. Two types of masters can be used. The first one summarizes the information between spots lists belonging to the same experiment and includes intensity considerations, while the second one summarizes the information resulting from different gels and does not include spot intensity and volume. Masters are written and known by the system as a spot list. This ability allows them to be handled just like spot lists and, using them, to perform all the actions available to the system. This approach is compared with a more classical one based on geometric corrections. The use of probability coefficients is also discussed and extended to their application to multiple matching and further utilization of masters.