bluuebunny/crossref_metadata_2025 huggingface dataset:
Superimposing two‐dimensional gels to study genetic variation in malaria parasites
Authors: Horgan Graham , Creasey Alison , Fenton Brian
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis is a valuable tool for studying genetic variation in the human malaria parasite, Plasmodium falciparum. It involves examining the positions of protein spots in gels produced from different isolates. Some spots have been seen to vary, while others have had a constant position in all isolates so far examined. These invariant spots provide a reference frame to compare variations in other spots. This paper discusses the usefulness of digital image handling, warping and superimposition in a personal computer environment. Rather than produce a fully automatic interpretation system, we show how the computer may be used as a tool for manipulating gel images, although interpretation of the gels' features remains with the human expert. Autoradiographs are scanned on a desktop scanner, and the images in digital form can be displayed on a monitor attached to a personal computer. The coordinates of the invariant spots on each of several gels are identified by the user. Each of the gels is then warped so that the invariant spots of all the gels coincide as closely as possible. The variable spots are then examined. We have used both affine warping transformations, which match the invariant spots as closely as possible, and thin plate spline transformations, which match them exactly. Colour superimposition proved a useful way of examining the gels.
A simple and rapid method for HLA‐DQA1 genotyping by polymerase chain reaction‐single strand conformation polymorphism and restriction enzyme cleavage analysis
Authors: Hayashi Tomonori , Seyama Toshio , Ito Takashi , Kusunoki Yoichiro , Hirai Yuko , Nakamura Nori , Akiyama Mitoshi
Published: May 2005

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)‐DQA1 locus is described. The polymorphic second exon of the HLA‐DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single‐strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, he eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA‐DQA1 genotypes should also be possible.
Microsequences of 145 proteins recorded in the two‐dimensional gel protein database of normal human epidermal keratinocytes
Authors: Rasmussen Hanne H. , Van Damme Josef , Puype Magda , Gesser Borbala , Celis Julio E. , Vandekerckhove Joël
Published: May 2005

Microsequencing of proteins recovered from two‐dimensional (2‐D) gels is being used systematically to identify proteins in the master human keratinocyte 2‐D gel database. To date, about 250 protein spots recorded in human 2‐D gel databases have been microsequenced and, of these, 145 are recorded in the keratinocyte database under the entry partial amino acid sequence. Coomassie Brilliant Blue‐stained protein spots cut from several (up to 40) dry gels were concentrated by elution‐concentration gel electrophoresis, electroblotted onto PVDF membranes and digested in situ with trypsin. Eluting peptides were separated by reversed‐phase HPLC, collected individually and sequenced. Computer search using the FASTA and TFASTA programs from Genetics Computer Group indicated that 110 of the microsequenced polypeptides shared significant similarity with proteins contained in the PIR, Mipsx or GenEMBL databases. Only 35 polypeptides corresponded to hitherto unknown proteins. Peptide sequences of all 145 proteins are listed together with their coordinates (apparent molecular weight and pI) in the keratinocyte database.
Human cerebrospinal fluid protein database: Edition 1992
Authors: Yun Miki , Wu Wei , Hood Lee , Harrington Michael
Published: May 2005

Two‐dimensional electrophoresis maps of human cerebrospinal fluid proteins are presented in the form of labeled images. 931 protein spots are identified in spinal fluid from a normal volunteer. Distinct spots that represent variants of the same protein, especially posttranslational modifications, are estimated to reduce the 931 different spots to < 200 different proteins. 248 spots of 29 protein groups have been identified and are indicated on enlargements of specific gel regions. The distribution of protein abundance, mass, charge and shape characteristics of these normal 931 spinal fluid spots are graphically profiled. Analysis of the shape parameter “vertical height: width ratio” reveals that a ratio > 3.5 correlates with glycoproteins, enabling their identification simply by image analysis. Proteins that are not present on the normal map, but appear in spinal fluid in patients with schizophrenia and Creutzfeldt‐Jakob disease are illustrated on additional maps.
Workshop on two‐dimensional gel protein databases
Authors: Simpson Richard J. , Tsugita Akira , Celis Julio E. , Garrels James I. , Werner Mewes H.
Published: May 2005

A workshop on two‐dimensional gel electrophoresis (2‐DE) protein database, organized by the Committee on Data for Science and Technology (CODATA) of the International Council of Scientific Unions Task Group on Biological Macromolecules, was held at the CODATA Secretariat in Paris on March 9, 1992. Eleven scientists from eight different countries represented various aspects of 2‐DE analysis – namely, cellular protein database development and protein microsequencing methodologies. The purpose of the workshop was to explore means of integrating the rapidly expanding body of information on 2‐DE resolved proteins from different laboratories. A major proposal emanating from the workshop was the establishment of an intermediary or “relational” 2‐DE gel protein database. This intermediary database, which would catalogue pertinent information on 2‐DE resolved proteins (experimental source, 2‐DE loci, biological information, etc.) could be an adjunct to, and accessed through, the existing international protein sequence databanks. It would function as a pointer for researchers to the individual 2‐DE protein databases where primary and more specialized 2‐DE data would be housed.
Two–dimensional polyacrylamide gel electrophoresis, with immobilized pH gradients in the first dimension, of barley seed proteins: Discrimination of cultivars with different malting grades
Authors: Görg Angelika , Postel Wilhelm , Baumer Max , Weiss Walter
Published: May 2005

The suitability of high‐resolution two‐dimensional gel electrophoresis for barley cultivar discrimination and for classification with respect to their malting properties was studied. Seed proteins of 14 barley cultivars with different malting qualities were extracted with urea/dithiothreitol/Nonidet P‐40 buffer and subjected to two‐dimensional gel electrophoresis with immobilized pH gradients in the first dimension (IPG‐DALT). The results of IPG‐DALT were compared to the protein patterns obtained by a standard technique, sodium dodecyl sulfate polyacrylamide gel electrophoresis of hordeins. Sodium dodecyl sulfate‐gel electrophoresis yielded seven different “B” and four different “C” hordein patterns; “A” and “D” hordein patterns were uniform in all cultivars tested. Four cultivars could be distinguished unequivocally, the others were classified into three groups containing between two and five cultivars. In contrast to these findings, IPG‐DALT yielded three different “A”, eight different “B”, four different “C” and two different “D” hordein patterns. When the “A”, “B”, “C” and “D” hordein patterns were combined, ten cultivars exhibited unique hordein patterns whereas the remaining ones were classified into two groups containing two cultivars each. Moreover, when albumin and globulin proteins were used for evaluation in addition to the hordeins, all cultivars could be discriminated by IPG‐DALT. IPG‐DALT, performed on small‐scale and/or ready‐made gels, proved to be an ideal complementary system to one‐dimensional electrophoretic methods for routine seed testing purposes because of its speed, reliability, and simplicity. IPG‐DALT was also applied to study the relationship between the different polypeptide patterns and the malting quality. Although cultivars with identical one‐dimensional protein patterns but different malting quality could be successfully differentiated by IPG‐DALT, a direct correlation between specific protein spots or protein patterns to the malting quality was not found within the cultivars tested.
Monitoring by two‐dimensional electrophoresis somatic embryogenesis in leaf and petiole explants from Vitis
Authors: Gianazza Elisabetta , De Ponti Paola , Scienza Attilio , Villa Pierluigi , Martinelli Lucia
Published: May 2005

A two‐dimensional electrophoretic analysis of the total proteins was carried out in Vitis rupestris as model system in order to characterize the different developmental stages – from callus to plantlets – of somatic embryogenesis events in the grapevine. The patterns of callus, embryogenetic callus, somatic embryos and plantlets derived from leaf and petiole explants were compared. Each differentiation step was characterized by specific peptide spots.
The use of immobilized pH gradients for the detection of human polymorphisms in the forensic identification of bloodstains
Authors: Muñoz‐Barús Ignacio , Carracedo Angel , Pascali Vincenzo L.
Published: May 2005

Some polymorphic proteins (α1‐antitrypsin, orosomucoid, transferrin, group specific component, plasminogen) and enzymes (phosphoglucomutase, acid phosphatase, estrase D) were determined in bloodstain extracts by isoelectric focusing with carrier ampholytes (CA) and with immobilized pH gradients (IPGs) rehydrated with CA. IPGs yield superior results for typing of genetics markers in bloodstains since phenotypes are better distinguished and the bands are straighter and sharper in the presence of contaminants. Also, the sensitivity of IPGs with CA is similar to isoelectric focusing (IEF) with CA. A new variant, ACP*B1, found in Negroid west African populations and not found in Caucasians is described. Such a variant can only be determined by IPGs since its isoelectric point (pI 5.95) is close to that of the ACP*B (pI 6.05) variant.
Rapid and simple method for the identification of apolipoprotein E isomorphic phenotypes from whole serum
Authors: Kohlmeier Martin , Drossel Hans‐Joachim , Sinha Pranav , Köttgen Eckart
Published: May 2005

For the identification of apolipoprotein E isomorphic phenotypes, fresh or thawed serum was analyzed without prior delipidation or other pretreatment. Using 5% polyacrylamide gels with a 40 mm interelectrode distance, the isoforms were separated by isoelectric focusing in immobilized pH gradients ranging from pH 5 to 6.5, and transferred onto polyvinylidene difluoride membranes by contact blotting for 1 h. The apolipoprotein E isoforms were identified following immunostaining. The electrophoresis required less than 2 h and the entire procedure could be completed within 6 h.
Microheterogeneity of apolipoprotein D as revealed by electroblotting following isoelectric focusing in Immobiline DryPlates
Authors: Holmquist Leif
Published: May 2005

The microheterogeneity of apolipoprotein D was examined by a procedure involving, in sequences: (i) electrophoresis in an immobilized pH 4–7 gradient in an Immobiline DryPlate‐polyacrylamide gel supplemented with Ampholine pH 5–7, (ii) covering of the gel with sodium dodecyl sulfate‐containing agarose, (iii) electroblotting onto a polyvinylidene difluoride membrane and (iv) immunological identification. Seven isoforms were obtained with partially purified apolipoprotein D. Using this technique the apparent pI values at 15°C for the isoforms were 4.57, 4.67, 4.78, 4.83 and 5.95, 6.06 and 6.19 (SD ± 0.05 for all). Direct staining of the Immobiline DryPlate could not reveal the isoforms of partially purified apolipoprotein D.
Modified silver staining for immobilized pH gradients
Authors: Rabilloud Thierry , Brodard Virginie , Peltre Gabriel , Righetti Pier Giorgio , Ettori Claudia
Published: May 2005

Silver development of gels containing an immobilized pH gradient has proved difficult so far because the bonded buffers (especially the tertiary amino acrylamido derivatives) tend to adsorb silver ions with a resultant heavy background of increasing darkness from the anode to the cathode. We report a variant of silver staining in which thiosulfate is used twice: (i) prior to silver impregnation, at the millimolar level, to enhance sensitivity, and (ii) during development, at the micro‐molar level, to decrease the background.
High resolution two‐dimensional gel analysis of proteins in the central nervous system of larvae of Drosophila melanogaster
Authors: Assigeo Rosa , Santarén Juan F.
Published: May 2005

An improved method of high resolution two‐dimensional gel electrophoresis was used to study the patterns of protein synthesis in the central nervous system (CNS) of late instar larvae of Drosophila melanogaster. A small number of CNSs was radiolabeled with a mixture of 14C‐labeled amino acids or with [35S]methionine, and the pattern of labeled proteins was analyzed. One thousand and forty‐five polypeptides, 800 acidic (IEF) and 245 basic (NEPHGE), from the CNS of several wild‐type strains have so far been separated and cataloged. All these polypeptides were numbered and compared with our catalog of polypeptides from wing imaginal discs, and quantitative or qualitative changes were detected in more than 23% of the polypeptides. When comparing patterns of label of CNSs we found small quantitative differences in the rate of synthesis between individuals of the same strain, not due to sexual differences, and a minute number of quantitative and qualitative differences between groups of individuals of different strains.
Errors in sizing bands of hypervariable DNA profiles on autoradiograms: Are they Gaussian?
Authors: Pascali Vincenzo L. , Moscetti Alessandra , Dobosz Marina , Pescarmona Marina , d'Aloja Ernesto
Published: May 2005

The accuracy of procedures for sizing hypervariable restriction fragments by Southern blot analysis (SBA) has been tested under three different experimental conditions: (i) intrablot serial analyses: three heterozygous DNA profiles were tested 14 times each in the same gel electrophoresis; (ii) intralaboratory analyses: we replicated three profiles (six autoradiographic bands) in over 100 SBA experiments; (iii) interlaboratory analyses: 15 serial measurements produced in a recent collaborative study (Forensic Sci. Int. 1991, 49, 1–15) were taken into account. In these three cases, a typical U‐shaped correlation curve between molecular size and coefficient of variation was found. We explain decrease of accuracy at both extremities of the gels in terms of: (i) enlarged shapes of bands and mean electrophoretic resolution at the cathode; (ii) diffusion and blurring of bands at the anodal edge. The three populations of data were subjected to a χ2 test and to the Kolmogorov‐Smirnov test in‐order to verify their compliance with normal distribution. Twenty‐three out of 27 tests indicated no significant deviation from the assumption of Gaussian distribution. We recommend the adoption of tests for normality to validate the use of symmetric confidence intervals for calculating gene frequencies and asserting a match between adjacent bands.
Analysis of collagen type III by uninterrupted sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotting: Changes in collagen type III polymorphism in aging rats
Authors: Takasago Teiichi , Nakamura Kazuyuki , Kashiwagi Shiro , Inoue Shin‐Ichi , Ito Haruhide , Takeo Kazusuke
Published: May 2005

A new method of type III collagen analysis by uninterrupted sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) combined with immunoblotting was developed. The electrophoresis was carried out with gels containing 4 M urea. A negatively charged reducing agent, thioglycolic acid, was added to the running buffer of the cathodic reservoir between 15 and 20 min after Bromphenol Blue (BPB) migrated to the top of the separating gel, to reduce interchain disulfide binding of the collagen. The polymorphic type III collagens, i.e., an α‐chain derived from a trimer [α1(III)]3 with interchain disulfide bonds but without covalent cross‐links, α1(III), a β‐chain with covalent cross‐links, β(III), or an α‐chain released from a trimer without reduction of the disulfide bonds, α*1(III), were identified by immunostaining and quantified by densitometry. Using this method, changes in collagen type III polymorphism with aging were examined in the aorta, brachial artery, and skin of rats. The total quantity of collagen type III decreased with aging in all tissues. β(III) was the major component in the aorta and brachial artery, but α1(III) was the major component in the skin. With increasing age from 3 to 60 weeks, the ratio of β(III) to α1(III), which is correlated with the extent of covalent cross‐linking, showed a steep increase in the aorta but only a slight increase in the skin and it remained almost constant in the brachial artery. The ratio of α*1(III) to α1(III), which is correlated with the extent of degradation, increased steeply from 12 weeks to 60 weeks in the aorta; in the brachial artery and the skin, however, it increased up to the 22nd week and was, thereafter, followed by almost constant values. These findings are characteristic of the profile of collagen type III metabolism in each of these tissues with aging.
Analysis of human tear proteins by two‐dimensional electrophoresis
Authors: Mii Seijiro , Nakamura Kazuyuki , Takeo Kazusuke , Kurimoto Shinji
Published: May 2005

Human tear proteins in the conjunctival sac were separated on the basis of the differences in their isoelectric points and molecular weights using micro two‐dimensional electrophoresis combined with immunoblotting. The two‐dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis were compared with those from normal individuals. We also measured integrated intensities of seven protein spots, lactoferrin (LF), albumin and five specific tear proteins (STP), to examine differences in the amounts of these proteins in tears from normal individuals of different sexes. In the tears from patients with conjunctivitis, secretory immunoglobulin A (IgA), LF and STP spots were stained more weakly, whereas the albumin spot was stained more strongly as compared with those from normal individuals. Furthermore, haptoglobin and IgG spots appeared in the tears from patients with conjunctivitis. These were more prominent in the tears from patients with severe conjunctivitis. There were significant differences in the amounts of LF and two kinds of STPs in the different sexes. The amounts of these proteins were larger in females.
Purification of serum amyloid A and its isoforms from human plasma by hydrophobic interaction chromatography and preparative isoelectric focusing
Authors: Malle Ernst , Heß Horst , Münscher Gerhard , Knipping Gabriele , Steinmetz Armin
Published: May 2005

The present work was aimed at isolating human serum amyloid A, (SAA), an acute‐phase protein mainly complexed to high density lipoproteins, directly from human plasma without sequential ultracentrifugation of lipoproteins and subsequent delipidation of the apolipoprotein moiety. Hydrophobic‐interaction fast‐protein liquid chromatography on Octylsepharose, using stepwise gradient elution profiles under dissociating conditions, followed by fast‐protein liquid‐gel permeation chromatography on a Superdex TM75 column revealed a higher than 95% purity of isolated SAA. Further purification of SAA from coeluting apolipoproteins C and A‐II was achieved by preparative isoelectric focusing between pH 5–7 using a Rotofor apparatus. Separation of the main SAA isoforms, SAA1 (pI 6.5) and SAA1 des‐Arg (pI 6.0, lacking the N‐terminal arginine), was achieved by anion‐exchange fast‐protein liquid chromatography on a Fractogel EMD DEAE 650‐S column. The purity of the SAA1 and SAA1 des‐Arg isoforms, thus isolated, was checked by immunochemical techniques and amino acid analysis. With the described method various SAA isoforms can be isolated, purified and separated directly from human plasma/serum without prior ultracentrifugation.
A comparison between low background silver diammine and silver nitrate protein stains
Authors: Rabilloud Thierry
Published: May 2005

Several methods of silver staining of proteins after sodium dodecyl sulfate‐electrophoresis in polyacrylamide gels were compared. The most rapid methods were found to be less sensitive than more time‐consuming methods. Among the long methods, those using glutaraldehyde treatment of the gel and silver diammine complex as the silvering agent were found to be the most sensitive, at the expense of the use of a modified polyacrylamide matrix and higher silver concentrations.
Theory of band broadening for DNA gel electrophoresis and sequencing
Authors: Slater Gary W.
Published: May 2005

The problem of (thermal) band broadening during DNA gel electrophoresis is studied analytically and numerically using the reptation model. It is shown that the orientation of the end‐to‐end vector of the molecules leads to increased diffusion and even to molecular size‐independent band broadening. Thus, the Einstein relation between mobility and diffusion holds for only very small molecules. Also, “self‐trapped” molecules are predicted to give fuzzy bands, as observed experimentally. Finally, megabase molecules are discussed and the consequences of our predictions for the improvement of sequencing are considered.
Gel electrophoresis of an end‐labeled DNA I. Dynamics and trapping in constant fields
Authors: Défontaines Anne‐Dominique , Viovy Jean‐Louis
Published: May 2005

A theory for the gel electrophoresis of a flexible polyelectrolyte, bearing an uncharged bulky label or an uncharged section at one end, is presented. We first consider a gel that is fully permeable to the label: we calculate the degree of stretching of the polyelectrolyte and its mobility as a function of chain size, electric field and label friction. Various regimes are identified, and their “existence domains” are calculated. For increasing friction, we predict a transition from a mobility decreasing with chain size to a mobility increasing with chain size. Secondly, we consider the possibility that the label may get trapped at some locations of the gel, a situation relevant to a method of “trapping electrophoresis” recently proposed by Ulanovsky et al. for DNA sequencing. A molecular model for detrapping by thermally activated “backward reptation” is constructed and solved using the Kramers rateequation theory. Different closed analytical expressions and approximate scaling laws corresponding to different regimes of stretching and field strengths are predicted. The most striking result is a mobility which exponentially decreases past a critical size N, which decreases with increasing field. In the regime relevant to the experiments by Ulanowsky et al., we predict N ∼ E−2/3. The predictions are in good qualitative agreement with presently available experiments, but further experimental investigations are suggested.
Effects of anomalous migration and DNA to protein ratios on resolution of equilibrium constants from gel mobility‐shift assays
Authors: Senear Donald F. , Dalma‐Weiszhausz Dennise D. , Brenowitz Michael
Published: May 2005

Numerical resolution of binding constants from data generated by the gel mobility‐shift assay is dependent on the experimental resolution of the different ligation states of the DNA. Previously we showed that the populations of the intermediate ligation states at partial saturation with the protein ligand are extremely sensitive to cooperativity (Senear, D. F. and Brenowitz, M. J. Biol. Chem. 1991, 266, 13661–13671). This makes accurate gel mobility‐shift data extremely useful to the demonstration of cooperativity. However, the accuracy with which the intermediate ligation state populations are resolved has been questioned. Thus, two additional and related questions are now considered. First, what information is available if the intermediate ligation state populations are not used in the analysis of binding constants. Second, is accurate information obtained from those states under conditions of high DNA concentration. These questions are addressed by using the interactions of the λ cI repressor protein with the three site operator, OR, and the interaction of the E. coli GalR protein with the single site operator, OE. Both simulated and experimental data are analyzed. The results point to two conclusions. First, precise resolution of all macroscopic constants for binding of proteins to DNA is critically dependent on the intermediate ligation state populations; resolution is limited to at most two DNA sites if these states are not used in the analysis. Second, when the DNA and protein concentrations used in the titrations are comparable, the resolution of binding constants is extremely sensitive to experimental uncertainty in the macromolecule concentrations.
Electroblotting of proteins on agarose gel containing specific antibodies
Authors: Atmeh Ragheb F. , Abuharfeil Nizar M.
Published: May 2005

A method is described for electroblotting of proteins, separated by gradient polyacrylamide gel electrophoresis, onto an agarose gel matrix containing specific antibodies. Three proteins of different molecular weight, including human albumin, isolated and in plasma, human plasma transferrin and C3 complement were tested. Immunoblotting on agarose, compared with nitrocellulose, was quantitative and highly sensitive, with small amounts of protein (i.e., 100 pg) being detected. Moreover, albumin aggregates (i.e., dimer, trimer and tetramer) were blotted quantitatively in addition to the monomer, and their percentages were calculated. This method is sensitive, quantitative, reproducible, and includes fewer manipulations; furthermore, it is less expensive and does not require the use of toxic or carcinogenic agents.
Lane distortions in gel electrophoresis patterns
Authors: Starita‐Geribaldi Mireille , Houri André , Sudaka Pierre
Published: May 2005

Distortions effects were studied as a function of gel environment, apparatus design and buffer type. Outward lanes distortions pronounced in low conductivity buffers for both continuous buffer systems and stacking gels, were field strength dependent. In discontinuous buffer systems, the moving boundary of the Laemmli buffer system deformed depending on the environment. In the Michov buffer system, the high conductivity resolving gel surrounded by a low conductivity electrode buffer, displayed straight pathways until a field strength of 15 V/cm, permitting to obtain information about lanes distortions depending on the acrylamide matrix structure. A decreased ammonium persulfate concentration, e.g. to 0.03%, induced phase segregation in gels at low temperature during the run: a critical endpoint near 11°C for a gel in the Michov buffer system under conditions of electrophoresis was supposed. Gel collapse resulted in inward deviations in the lanes of the pattern and also faint bands in the zones where the gel was retracted. Besides this effect of catalyst concentration, the inwardly distorted pattern depended on temperature, field strength, solvent and hydrolysis due to gel aging. Amendments for overcoming lane distortions are suggested.
Electrotransfer of fixed phosphoproteins from pieces of dried polyacrylamide gel to small disks of nitrocellulose, nylon or polyvinylidene difluoride
Authors: Gonçalyes Carlos A. , Rodnight Richard
Published: May 2005

A simple method for the transfer of 32P‐labeled proteins from dried polyacrylamide gels to small disks of nitrocellulose, nylon or polyvinylidene difluoride (PVDF) is described. Gel pieces containing the desired phosphoprotein are rehydrated in buffer containing sodium dodecyl sulfate (SDS) and sealed in agarose in a glass tube over a supporting gel of polyacrylamide. Protein is transferred upwards through a discontinuous density gradient of SDS‐buffer and methanol to a disk of membrane sealed to the mouth of the tube with dialysis membrane. The method allows the concentration of a phosphoprotein present in several gel pieces to a single disk of immobilized membrane. Recovery of phosphoprotein was at least as good as obtained with conventional electroblotting. Application of the method to the analysis of the phosphoamino acid content of the astrocyte marker, glial fibrillary acidic protein, is described.
Differentiation of proteins in polyacrylamide gels by a modification of silver staining for the PhastSystem and a laser densitometer
Authors: Kierdorf Horst , Melzer Heinrich , Mann Helmut , Sieberth Heinz‐Günther
Published: May 2005

Nonspecific background staining of the gel matrix is the limiting factor in the differentiation of proteins using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. It is caused by nonspecific binding of silver ions in the gel matrix, either to nonprotein compounds or through the chemistry of the polyacrylamide gel itself. The resulting stain of the gel produces a stain baseline, making differentiation of protein bands with a laser densitometer difficult. We have therefore developed a modified silver staining method for the PhastSystem to reduce such nonspecific background staining. Apart from slowing the development program by lowering the temperature to 15°C and shortening the incubation time to 4 min, the essential step in the modification is the combined use of an EDTA and Tris‐acetate buffer solution which stops the reduction of silver ions. The reduced background staining leads to an improved detection of protein bands and virtually identical zero lines for the laser densitograms and the stain baselines. The total staining time is 91 min. All steps in the program are fully automated and continuous, employing the PhastSystem staining unit.
Microsequence analysis of the N‐terminally blocked proteins immobilized on polyvinylidene difluoride membrane by Western blotting
Authors: Hirano Hisashi , Komatsu Setsuko , Kajiwara Hideyuki , Takagi Yoshiaki , Tsunasawa Susumu
Published: May 2005

A technique has been developed for efficient deblocking and subsequent microsequencing of N‐terminally blocked proteins immobilized on a polyvinylidene difluoride (PVDF) membrane at the picomole levels. In this technique, proteins were first separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane by Western blotting. The electro‐blotted proteins with N‐terminal acetylserine or acetylthreonine could be deblocked on‐membrane by treatment with trifluoroacetic acid vapor and sequenced by a gas‐phase protein sequencer. Similarly, N‐formylated proteins could be deblocked on‐membrane in HC1 solution and then directly sequenced from the N‐terminal amino acid. Proteins with N‐terminal pyroglutamic acid were enzymatically deblocked by in situ pyroglutamyl peptidase digestion, and N‐acetylated proteins were also enzymatically deblocked with acylamino acid‐releasing enzyme (AARE) after on‐membrane digestion with trypsin to generate the N‐terminal peptide fragment. This tryptic digestion was required since AARE can remove the acetylamino acid only from a short peptide. Based on these four deblocking methods, we present a strategy for sequential deblocking and subsequent N‐terminal sequence analysis of N‐blocked protein immobilized on PVDF membrane.
Time‐resolved fluorometry in nucleic acid hybridization and Western blotting techniques
Authors: Diamandis Eleftherios P.
Published: May 2005

Time‐resolved fluorometry is now used extensively for immunological assays and to a lesser extent in other research areas. In this review I describe applications of time‐resolved fluorometry in nucleic acid hybridization and in blotting techniques, including Southern and Western blotting. Clearly, timeresolved fluorometry has potential for playing a major role in techniques other than immunological assays.
Protein blot assays specific for the discrimination of the centromere autoantigen, CENP‐A, from human cells
Authors: Billings Peter B. , Martinez Antigona , Haselby Jeanne A. , Hoch Sallie O.
Published: May 2005

The Western or protein blot has proven to be a valuable resource in detecting discrete, immunoreactive antigen targets associated with a variety of autoimmune diseases. As the roster of autoantigens has expanded, it has become increasingly common to tailor specific gel or blot conditions to a particular polypeptide antigen. Two such assays are reported here as applied to the fractionation and visualization of human centromere protein (CENP‐A), a centromere autoantigen associated with the rheumatic disease, systemic sclerosis. The centromere antigens are effectively solubilized in the presence of 1 M MgCl2 to allow for further purification. CENP‐A copurifies with the histone proteins, primarily H3 and H4. The two CENP‐A‐specific protein blot assays separate CENP‐A from the histone proteins and enhance CENP‐A immuno‐reactivity. The first assay is based on the use of acid‐urea gels with a Triton X‐100 concentration chosen to maximize separation of CENP‐A from all the histones. The second assay is based on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis to differentiate two very basic proteins of similar molecular weight, namely CENP‐A and histone H3. For each gel system, a selective choice of associated immunoblot parameters allows for the reproducible discrimination of the CENP‐A antigen.
The Pearson product‐moment correlation coefficient is better suited for identification of DNA fingerprint profiles than band matching algorithms
Authors: Häne Bernhard G. , Jäger Kathrin , Drexler Hans G.
Published: May 2005

A database of DNA fingerprint profiles from permanently established human and animal cell lines was prepared with a computer program originally designed for numerical taxonomy of bacteria. Identifications of cell line DNA profiles were performed, both by the Pearson product‐moment correlation coefficient and by band matching. Under the conditions used the Pearson product‐moment correlation coefficient was consistently more reliable.
Heterogeneity of carbonic anhydrase and 68 kDa neurofilament in nerve roots analyzed by two‐dimensional electrophoresis
Authors: Fujii Hiroshi , Nakamura Kazuyuki , Takeo Kazusuke , Kawai Shinya
Published: May 2005

The heterogeneity of carbonic anhydrase (CA) and 68 kDa neurofilament (NF) in nerve roots of Japanese monkeys (Macaca Macacus) was analyzed by two‐dimensional electrophoresis combined with immunoblotting. CA was detected as heterogeneous spots of type I and II having the same molecular weight (Mr) but different isoelectric point (pI), in the range of pH 4.5–5.5. CA had much higher concentrations in soluble fractions of dorsal roots than ventral roots but not in membrane‐bound fractions of either root. NF was detected as a single spot at the position of 68 kDa and pI 5.2 in soluble fractions of both roots, however, it was detected in the membrane‐bound fractions as heterogeneous spots having different molecular masses and pI in the range of 60–68 kDa and 5.0–5.5, respectively. The presence of heterogeneous CA type I and II at high concentrations in dorsal roots enables us to distinguish the sensory nerves from the motor nerves. Furthermore, the heterogeneity of NF in membrane‐bound fractions gives us information for understanding the dynamic assembly and the degradation of NF in nerve roots.
The human keratinocyte two‐dimensional gel protein database: Update 1993
Authors: Celis Julio E. , Rasmussen Hanne Holm , Olsen Eydfinnur , Madsen Peder , Leffers Henrik , Honoré Bent , Dejgaard Kurt , Gromov Paul , Hoffmann Hans Jürgen , Nielsen Morten , Vassilev Alex , Vintermyr Olav , Hao Junshan , Celis Ariana , Basse Bodil , Lauridsen Jette B. , Ratz Gitte P. , Andersen Annette H. , Walbum Else , Kjærgaard Inge , Puype Magda , Van Damme Josef , Vandekerckhove Joël
Published: May 2005

The master two‐dimensional gel database of human keratinocytes currently lists 3038 cellular proteins (2127 isoelectric focusing, IEF; and 911 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to post‐translational modifications. 763 proteins have been identified (protein, name, organelle components, etc.) and they are listed both in alphabetical order and with increasing SSP number, together with their Mr, pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore we have listed 176 proteins that have been microsequenced so far and that are recorded in this database. We also include synthetic images depicting some interesting sets of proteins identified so far; these include components of hnRNP's, proteasomes or prosomes, ribosomes, as well as assorted organelle markers, GTP‐binding proteins, calcium binding proteins, stress proteins, autoantigens, differentiation markers and psoriasis upregulated proteins. The aim of the comprehensive database is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and ultimately to pinpoint signaling pathways and components affected in various skin diseases, cancer included.
Influence of the electrode compartment separating membranes on continuous flow electrophoresis
Authors: Heinrich Jörn , Wagner Horst
Published: May 2005

The influence of the ion permeable diaphragms, separating the electrode compartments of continuous flow electrophoresis chambers from the separation space, are described in detail. A set of equations is derived from a simplified phenomenological model of the ion transport across media boundaries. These equations are useful to predict the magnitude of concentration changes near the membranes due to the electrophoretic ion transport. Adaption to any CFE geometry, membrane material and electrolyte system is possible with only a few measurements, which yield the transference number difference as a characteristic transport property of the membrane material for a special electrolyte. The validity of the equations is checked by experiments. Local electric field strength and local conductivity are measured with a potential gradient‐conductivity scanner developed in our laboratory. This device yields data on the dynamics of the concentration changes.
Human liver protein map: Update 1993
Authors: Hughes Graham J. , Frutiger Séverine , Paquet Nicole , Pasquali Christian , Sanchez Jean‐Charles , Tissot Jean‐Daniel , Bairoch Amos , Appel Ron D. , Hochstrasser Denis F.
Published: May 2005

This publication updates the reference human liver protein map. By microsequencing, 27 spots or 34 polypeptide chains were identified. The most abundant polypeptides detected on the silver stained liver map were key elements in major hepatic biochemical pathways. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing the 1993 reference human liver SWISS‐2DPAGE database. SWISS‐2DPAGE and the SWISS‐PROT protein sequence databases are closely linked together through the use of common accession numbers.
How far have we progressed toward automated electrophoresis in sieving media of the twenty‐first century?
Authors: Chrambach Andreas
Published: May 2005

The automation of electrophoresis in polymeric sieving media requires (i) an objective definition of the conditions (the polymer, its concentration, solvent, buffer, pH, ionic strength, temperature) under which a particular separation proceeds most effectively; (ii) apparatus capable of zone detection, acquisition by computer, evaluation (migration distance, zone width and area) and a printout of the number of components, their size and net charge, and the polymer conditions under which each component separates most effectively from its two neighboring zones. Both of these prerequisites of automation have been met to a first approximation at this time and, after further maturation, assembly and streamlining should be able to fill the need of the coming century for a more efficient, nonarbitrary and cost‐effective mode of macromolecular and cellular particle separation. (i) The realization of the qualitative equilivalence of polymer solutions and gels had greatly increased our options in the choice of sieving media. That choice can be made objectively by correlating separation efficiency (S), particle size (R) and intrinsic viscosity (ηo) of the polymer. (S) is a function of the slope, KR (R), of the Ferguson plot [log(mobility) vs. polymer concentration], or with nonlinear plots (DNA, agarose) KR(R,T). KR is at present most easily derived from transverse pore gradient gels or by conducting capillary zone electrophoresis (CZE) at multiple polymer concentrations. Pore gradient CZE appears promising. CZE also defines the free mobility unequivocally. Computer programs exist to generate KR from migration distances (times), and optimal S and polymer concentration for a particular R from KR. Optimal S can be related objectively with ηo to define, as a first‐order approximation, the optimal polymer for the separation within a narrow R‐range. (ii) Apparatus with automated detection of zones and acquisition of migration distances has become commercially available (e.g. Applied Biosystems, Du Pont, LabIntelligence). One of these scans multiple lanes and can be interfaced to compute KR and S. Thus, all elements necessary for automated electrophoretic separation under optimally effective separation conditions are available in rudimentary form. Their development, assembly, integration and streamlining should be feasible prior to the onset of the next century.
Electrophoretic light scattering
Authors: Takagi Toshio
Published: May 2005

Under a microscope, electrophoretic mobilities of visible particles can be determined in a free solution without any boundary or zonal separation. Electrophoretic light scattering based on the Doppler velocimetry can be taken as a nanometer‐scale version of the above traditional electrophoresis. An electrophoretic light scattering apparatus was developed to enable mobility determination of proteins and surfactant micelles which have been outside the range of the electrophoresis.
Electrophoretic artifacts arising from the use of thiol‐containing reagents
Authors: Gianazza Elisabetta , De Ponti Paola
Published: May 2005

Thiol reagents migrate as a curtain behind the salt front when loaded with the sample solution onto disc‐electrophoresis gels. In immobilized pH gradients (IPG) the same compounds are driven by electrophoresis and electroosmosis from the alkaline to the neutral and acidic regions of the gradients. In either case, a dose‐dependent sideways spreading results in spurious reduction between adjacent lanes if samples with and without reducing agent are loaded side by side.
On the use of dimensionless parameters in acid‐base theory. V. Buffers composed of binary mixtures of monovalent weak acids and bases
Authors: Rilbe Harry
Published: May 2005

A general theory for buffers consisting of one weak acid and one weak base is developed, particular interest being devoted to the special case of doubly weak salts. The pH course as it varies with the acid/base concentration ratio is presented in equations and graphs. The influence of ΔpK′ = pK′b−pK′a, which can be negative as well as positive, is discussed and visualized in graphs. The buffer capacity is deduced as a function of pH. The buffer range is, at a maximum, 2.67 times that of a monovalent weak protolyte for ΔpK′ = 2.7 pH units. The buffer capacity curve has two maxima for ΔpK′ values bigger than 0.77. For negative ΔpK′ values the buffer capacity curve has only one maximum, and the buffer range is not better than that of a monovalent protolyte. A general equation for the ionic concentrations is deduced, and as a corollary the degree of hydrolysis of the doubly weak salt is obtained as a function of ΔpK′. For solutions of this salt, the relation between concentration and pH is elucidated by equations and graphs.
Application of an improved density gradient electrophoresis apparatus to the separation of proteins, cells and subcellular organelles
Authors: Tulp Abraham , Verwoerd Desirée , Pieters Jean
Published: May 2005

A DGE apparatus, made of Perspex, consisting of a separation column (5 × 2.2 cm) and containing a 0–4% linear Ficoll density gradient, was constructed. Only 2.5 cm of the column were used for high resolution separations. A specially designed removable top cone permitted precise gradient introduction, thin sample layering (0.3–1 mm) and precise fractionation after electrophoresis. A bottom circular palladium anode (nongassing) was separated hydrodynamically but not electrically from the density gradient by a cationpermeable membrane. A top circular platinum cathode caused negatively charged particles to migrate upwards (levitation). Thin sample layering permitted short separation times (30–60 min) at only 3 V/cm (10 mA). As for proteins, glycoforms of α1‐antitrypsin were separated as well as isoenzymes of β‐hexoseaminidase. Furthermore, separation of transferrin (Tf) from the putative Tf‐receptor complex was effectuated. The device was equally suitable for the separation of Megadalton proteins (mucins). Artificial mixtures of intact erythrocytes (rat, rabbit, human) were separated with high resolution. About 107 cells (of 100 μm3 cell volume) could be loaded onto the device. Crude microsomes from the human melanoma cell line Mel JuSo were separated after brief trypsin treatment within 38 min at 10 mA. Ratios of the migration velocities of the constituent organelles were: late endosomes (LE) : lysosomes (L) : Golgi (G) : early endosomes (EE) = 1 : 0.94 : 0.77 : 0.55 and under slightly different conditions LE : L : G : endoplasmatic reticulum (ER) : plasma membrane (PM) = 1 : 0.87 : 0.64 : 0.58 : 0.49.
A fast spot segmentation algorithm for two‐dimensional gel electrophoresis analysis
Authors: Wu Yecheng , Lemkin Peter F. , Upton Kyle
Published: May 2005

An important issue in the automation of two‐dimensional gel electrophoresis image analysis is the detection and quantification of protein spots. A spot segmentation algorithm must detect, define the extent of, and measure the integrated density of spots under a wide variety of actual gel image conditions. Besides these functions, the algorithm must be memory efficient to be able to process very large gel images and do this in a reasonable amount of computation time on low‐cost computers, such as workstations and personal computers. We have developed a fast spot segmentation algorithm, extending the GELLAB‐II segmenter, which extracts spots in a single raster scanning pass through the gel image. The performance analysis of the algorithm will be given in the paper as well as a discussion of the algorithm.
Bronchoalveolar lavage fluid proteins in human lung disease: Analysis by two‐dimensional electrophoresis
Authors: Lenz Anke‐Gabriele , Meyer Barbara , Costabel Ulrich , Maier Konrad
Published: May 2005

Proteins of human bronchoalveolar lavage fluids, obtained by washing the epithelial lining fluid of the lungs with phosphate‐buffered saline, were analyzed by two‐dimensional electrophoresis under denaturating and reducing conditions. The two‐dimensional pattern of bronchoalveolar lavage fluid proteins of healthy volunteers (controls) were compared with those of patients with idiopathic pulmonary fibrosis, sarcoidosis, and asbestosis. Particular interest was paid to the proteins present in minor amounts mainly in the low molecular weight region of the gels. Marked changes in single protein spots were observed. In idiopathic pulmonary fibrosis the spot intensity of the surfactant‐associated protein, SP‐A, showing isomeric forms both in charge and in molecular weight, was markedly decreased. In sarcoidosis, the immunoglobulins (IgG, IgA) and a group of protein spots at an isoelectric point of 4.5–5.0 and a molecular mass of 55 kDa were increased. An additional spot appeared at an isoelectric point of 4.5 and a molecular mass of 12 kDa. In particular in asbestosis, but also in some cases of idiopathic pulmonary fibrosis and sarcoidosis, the number and intensity of low molecular weight proteins were increased strongly.