bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of mouse brain proteins after two‐dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis
Authors: Eckerskorn Christoph , Jungblut Peter , Mewes Werner , Klose Joachim , Lottspeich Friedrich 
Published: May 2005

AbstractTwo‐dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time‐consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low μg range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two‐dimensionalelectrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R‐250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N‐terminal sequence determination.
Immunoblotting techniques with picogram sensitivity in cerebrospinal fluid protein detection
Authors: Nespolo Angelo , Bianchi Giorgio , Salmaggi Andrea , Lazzaroni Monica , Cerrato Dario , Tajoli Livia Malesani 
Published: May 2005

AbstractAgarose isoelectric focusing followed by blotting with nitrocellulose, nylon or polyvinylidene difluoride membranes, and immunochemical detection of cerebrospinal fluid IgG with various combinations of antisera, was evaluated. Polyvinylidene difluoride proved to be an easy‐to‐handle and reliable membrane for protein blotting. Among immunochemical visualization reactions, the most sensitive employed biotinylated goat anti‐human IgG followed by streptavidin colloidal gold conjugate and silver enhancement in 20% w/v urea, allowing a sensitivity of less then 1 picogram IgG/band.
Systematic analysis of the total proteins of a mammalian organism: Principles, problems and implications for sequencing the human genome
Authors: Klose J. 
Published: May 2005

AbstractHigh‐resolution two‐dimensional electrophoresis (2‐DE) has reached a technological level that allows us to resolve most of the numerous unknown protein species of a mammalian organism if appropriate strategies are used. We will discuss the problems of classification and characterization of proteins and propose a systematic approach to the analysis of the total protein complex. Both a comprehensive as well as a pragmatic approach towards systematic analysis have been considered. A “complex protein database” is suggested and considered with regard to various uses. A systematic analysis of the mouse proteins has been started and some of the preliminary results are summarized here. In particular, genetic properties of the proteins were investigated and are presented in order to demonstrate the significance of a systematic analysis of proteins for research and practical application (e. g. mutagenicity testing). A concept is presented for sequencing the coding DNA of mouse and man, starting with a systematic analysis of mouse proteins and then using two recently developed methods ‐ microsequencing of proteins from spots of 2‐DE protein patterns, and utilization of the relatively short N‐terminal sequences obtained – to produce the corresponding cDNA's of these proteins.
The migration anomaly of DNA fragments in polyacrylamide gels allows the detection of small sequence‐specific DNA structure variations
Authors: Diekmann Stephan 
Published: May 2005

AbstractCurved DNA fragments have a reduced electrophoretic mobility in polyacrylamide gels. The retardation in gels is extremely sensitive to small structural variations which influence the DNA helix axis. This gel assay can also be used to detect very small structural variations in DNA sequences which are not curved: The noncurved seguences of interest can be combined with curved stretches in phase with the helix turn. Using such sequence constructions, even subtle influences on the DNA helix axis can be detected. Experiments of this kind allow the determination of a relative order of sequence‐specific DNA twist and wedge angles.
Computer‐assisted evaluation of polydisperse two‐dimensional gel patterns of polysaccharide‐protein conjugate preparations with regard to size and net charge
Authors: Tietz Dietmar , Chrambach Andreas 
Published: May 2005

AbstractNative Hemophilus influenzae polysaccharide‐protein conjugate particles were analyzed by a two‐dimensional agarose electrophoresis procedure. In view of their preparation by random chemical crosslinking, the conjugates necessarily exhibit a polydisperse two‐dimensional gel pattern which varies depending on the conditions of the particular preparation. The polydisperse patterns were interpreted with regard to the size and surface net charge density of the conjugate on the basis of the extended Ogston model. Data processing was performed by a new program, designated ZWEIDI.DO, written in the language of M‐LAB (modeling laboratory). The program computes particle and gel fiber specific parameters from the positions of standards and unknown(s) on the two‐dimensional gel using a simultaneous linear least‐square curve fitting routine. Based on these calculations, the program serves to compute a nomogram of iso‐size and iso‐free‐mobility profiles. Superimposing these profiles on the gel patterns, the size and free mobility range of the polydisperse conjugate mixtures is obtained. Potentially, the procedure could serve as a tool for quality control in the production of conjugates as vaccines and for the physical characterization of polydisperse subcellular particles and vesicles.
Consecutive radiofluorography and silver staining of two‐dimensional gel electrophoretograms: Application in determining the biosynthesis of serum and tissue proteins
Authors: Kuhn Lotte , Kettman Jack , Lefkovits Ivan 
Published: May 2005

AbstractThe methodology for conventional radiofluorography of two‐dimensional gels, followed by rehydration of the gel and subsequent silver staining, is described. The image obtained by radiofluorography is referred to as biosynthetic image, and the image obtained by silver staining as constitutive image. Since the two images are already in close register (the same gel), reliable identification of polypeptides by the two different assays is possible, and the comparison provides valuable information on the catabolism of each entity. The utility of this procedure is illustrated in experiments involving a labeling with L‐[35S] methionine of an entire mouse. Both serum and tissue samples were analyzed by two‐dimensional gel electrophoresis with the aim of determining several categories of polypeptides in terms of their biosynthetic rates and their composition.
Two‐dimensional electrophoresis aided by personal computer analysis for screening of mutant proteins in inherited diseases
Authors: Yang Rei‐Cheng , Suzuki Yoshiyuki , Tsuji Akihiko 
Published: May 2005

AbstractA rapid and reproducible method of two‐dimensional electrophoresis was developed for screening of abnormal proteins expressed in fibroblasts from patients with inherited diseases. After silver staining, the electrophoresis gel was subjected to semiautomatic digitizer ‐ personal computer analysis: scanning with an image sensor video camera connected to a digitizer, followed by quantitative determination and statistical analysis with a personal computer. The protein spots analyzed by this method showed quantitative variations of various degrees, particularly in 2 of 247 spots examined. Seven spots were not always detected in control and pathological cells in this study. Slight variations in molecular weight were observed in 3 different spots.
Important parameters in semi‐dry electrophoretic transfer
Authors: Jacobson Gunilla , Kårsnäs Per 
Published: May 2005

AbstractThe efficiency of semi‐dry electrophoretic transfer after sodium dodecyl sulfate (SDS)‐electrophoresis using PhastGel® media was investigated in a model system using three isotope labelled proteins. To give a full picture of the blotting process the amount of protein present in the gel, membranes, and filter papers was determined after different transfer times. The influence of the transfer buffer, commonly used additives such as methanol and SDS, and several different immobilizing matrices was investigated. Soybean trypsin inhibitor, bovine serum albumin, and ferritin were used as model proteins to study the effect of size on transfer efficiency. Basically, all three stages of the blotting process decide the result; the elution of protein from the gel, the immobilization of protein to the membrane, and the loss of material from the membrane during transfer. A theoretical explanation for the observed poor binding to a second membrane is discussed. Our results show that the buffer composition has little influence on the efficiency of transfer from the gel, but can be significant to the binding capacity of the membrane. In all experiments performed, there was never one moment during the transfer when all protein was eluted from the gel and simultaneously still bound to the membrane. The highest recovery in the membrane was obtained at different time intervals for different proteins. This indicates that quantitative transfer procedures cannot be generalized. However, obtaining an optimal method for reliable quantification of a specific protein or group of proteins is possible. For general protein staining of nitrocellulose and polyvinylidene difluoride membranes, a highly sensitive silver staining method requiring only 15 min has been used.
Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of Coomassie Brilliant Blue and silver staining
Authors: Warlow Robert S. , Bernard Claude C. A. 
Published: May 2005

AbstractOptimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol‐acetic acid‐formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.
Use of two‐dimensional gel electrophoresis and autoradiography as a tool in cell biology: The example of the thyroid and the liver
Authors: Lecocq Raymond , Lamy Françoise , Dumont Jacques E. 
Published: May 2005

AbstractDifferent applications of two‐dimensional gel electrophoresis and the research strategies that this methodology allows, with examples drawn from our own work on thyroid and liver cells, are described.
Experimental and theoretical description of the isotachophoretic behavior of serum albumin
Authors: Thormann Wolfgang , Mosher Richard A. 
Published: May 2005

AbstractThe isotachophoretic behavior of serum albumin is examined for three anionic and one cationic electrolyte systems by (i) computer simulation, (ii) capillary isotachophoresis (ITP) and (iii) continuous flow ITP. The theoretical relationship between pH of the leading electrolyte and the steady state protein plateau concentration is presented for one of the anionic systems. With leading ion concentrations of the order of 10 mm, experimental protein plateau concentrations of 1.3–2.3 % w/v are obtained. The computer predictions are approximately half these values.
Numerical analysis of sodium dodecyl sulphatepolyacrylamide gel electrophoretic protein patterns for the classification, identification and typing of medically important bacteria
Authors: Costas Menelaos 
Published: May 2005

AbstractA numerical analysis of one‐dimensional sodium dodecyl sulphate polyacrylamide gel electrophoretic protein patterns of three separate bacterial groups is described. Similarity between patterns was estimated using a correlation coefficient and is represented diagramatically by the unweighted pair‐group with arithmetic averages method of clustering. Protein patterns were scanned using a laser densitometer interfaced directly to a microcomputer, where calculations were performed semiautomatically with specially written software. A method for the normalization of patterns from different gels is utilized. Whole cell protein patterns clearly distinguished strains representing various groups of Achromobacter. Similarly the two biovars of the Group EF‐4 bacteria could be separated after analysis of partial „background patterns”︁ as could the different species of Providencia. In addition, whole patterns could be used to type the various strains of P. rettgeri. These examples serve to illustrate the power, versatility and potential of the technique when applied to problems of classification, identification and typing of bacteria of medical interest. Its advantages and disadvantages, when compared to other available electrophoretic methodologies, are discussed, as is the future application of such technology to the hospital laboratory.
Blotting efficiency investigated by using two‐dimensional electrophoresis, hydrophobic membranes and proteins from different sources
Authors: Jungblut Peter , Eckerskorn Christoph , Lottspeich Friedrich , Klose Joachim 
Published: May 2005

AbstractPurification and chemical characterization of protein may be achieved by combining two‐dimensional electrophoresis (2‐DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2‐DE pattern as possible. Under optimized conditions, blotting efficiencies between 60 % and 100 % were obtained for five marker proteins; the mean values from four regions of a 2‐DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60 % and 79 %. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patters, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.
An improved scheme for the identification of antigens recognized by specific antibodies in two‐dimensional gel electrophoresis and immunoblotting
Authors: Shen Horng‐Der , Choo Kong‐Bung , Lin Win‐Ling , Lin Run‐Yee , Han Shou‐Hwa 
Published: May 2005

AbstractThis paper describes an improved scheme for the identification of antigens in crude extracts recognized by specific antibodies when analyzed by a combination of two‐dimensional gel electrophoresis and immunoblotting. First, protein components in gels are electrophoretically transferred to a polyvinylidene difluoride membrane which does not shrink or change dimensions in organic solvents. The efficiency of transfer and the localization of sample proteins on the membrane are checked and recorded by staining the blotting membrane with Fast Green FCF and recording the profile on a transparency. After blocking and the immunoassay, the results are recorded by photography. The sites of immune reaction are marked and the same membrane is restained briefly with Coomassie Brilliant Blue R‐250 for the protein profile. Thus antigens in complex mixtures, recognized by antibodies of interest, can easily be identified from the restained membrane. If the whole protein profile is not well demonstrated, when used in combination with the profile recorded on the transparency, spots appearing on the restained membrane can still be used as useful landmarks in the final unequivocal antigenic identification. This improved scheme circumvents problems arising from membrane shrinkage and difficulties in accurately matching immunoreactive spots by conventional procedures and thus provides an accurate, simple and fast approach in the identification of antigens after immunoblotting.
Development of a database of amino acid sequences for human colon carcinoma proteins separated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Ward Larry D. , Hong Ji , Whitehead Robert H. , Simpson Richard J. 
Published: May 2005

AbstractThe tandem use of preparative two‐dimensional polyacrylamide gel electrophoresis (2‐DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R‐250, and selected protein spots were excised and subjected to Edman degradation. Our résults demonstrate that overall sequence yields in the 3–20 pmol range can be achieved on protein spots from four identical 2‐DE gels; approximately 150–200 μg of total protein was applied to a single 2‐DE gel. An approximate two‐fold increase in sensitivity of phenylthiohydantoin‐amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin‐amino acid derivative. N‐Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of protein of known structure listed in the available protein sequence databases. Approximately 40 % of the electroblotted proteins did not yield N‐terminal sequence information, presumably because they had blocked N‐termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2‐DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one‐dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.
Influence of carbohydrate moieties of human serum transferrin on the determination of its molecular mass by polyacrylamide gradient gel electrophoresis and staining with periodic acid‐Schiff reagent
Authors: Riebe Dieter , Thorn Werner 
Published: May 2005

AbstractThe influence of carbohydrate moieties of transferrin (Tf) on the determination of its molecular mass (MM) by polyacrylamide gradient gel electrophoresis (PAGGE) was investigated. Iron‐free native human serum transferrin (Tf) of 99% purity and partly or completely carbohydrate‐ and N‐acetylneuraminic acid (NANA)‐free molecule forms were analyzed. The MM differences before and after enzymatic cleavage were found not to agree with the theoretical difference. From amino acid and carbohydrate analysis the MM of Tf was determined to be 79 570 Da whereas by denaturing and nondenaturing PAGGE MM of 77 000 Da ± 1000 Da were found. After enzymatic cleavage of the two carbohydrate chains of Tf the difference between the calculated MM and the value reported in literature increased to 7000 Da (nondenaturing PAGGE) and 9200 Da (denaturing PAGGE). Following enzymatic cleavage of the 4 NANA molecules (MM 1237, Da) we obtained the relatively largest difference between the value given in the literature and that determined by PAGGE, namely MM 3300 Da on nondenaturing and 4000 Da on denaturing PAGGE. The differences due to the removal of the other carbohydrates were negligible. In addition we tested the periodic acid‐Schiff reagent to stain iron free Tf, containing different carbohydrate residues. The shortest carbohydrate moiety necessary for Tf staining corresponds to two identical carbohydrate chains of the structure (Asn)‐GlcNAcGlcNAc‐α‐Man‐(α‐Man‐)‐α‐Man.
Detection of conformational and net charge differences in DNA‐protein complexes by quantitative electrophoresis on polyacrylamitie‐agarose copolymer gels
Authors: Orbán Lászó , Chrambach Andreas , Zwieb Christian , Adhya Sankar L. 
Published: May 2005

AbstractThe Galactosidase repressor (GalR) of Escherichia coli modulates the expression of the gal operon by binding to two DNA operators, OE and O1. The OE and O1 elements are 16 by pallindromic DNA sequences, differing in four of the base pairs (Fig. 1). OE and O1 DNA fragments, both free and complexed with repressor, were analyzed by “quantitative gel electrophoresis”. By the criteria of that method, applied to the linear Ferguson plots of both DNA fragments and the linear ranges of those of the DNA‐GalR complexes, it was shown that the apparent size of DNA increases upon repressor binding. Moreover, this size increase is greater for the complex with the O1 operator than for the complex with the OE operator in the case that GalR is located in the center of a 155 bp DNA fragment. This is not the case when GalR is located in a peripheral position. By contrast with their size differences, the centrally located GalR‐O1 and GalR‐OE complexes appear to possess indistinguishable net surface charge densities as judged from the intercepts with the mobility axis. The larger size of the complex with centrally located O1 fragment, as compared with that bearing the OE fragment, is interpreted as being due to bending of the DNA‐protein complex, since an authentically bent fragment of a plasmid with bent upstream activator sequence also exhibits a larger slope of the Ferguson plot, and thus the larger size, than predicted on the basis of its DNA chain length (bp). Thus, the apparently larger size of the GalR‐O1 complex at the center of DNA, compared to that of the GalR‐OE complex, supports the previous conclusion that the Gal repressor induces a larger degree of bending in O1 DNA than in OE DNA. DNA fragments bearing OE and O1 when complexed with the Gal repressor show convex Ferguson plots at polyacrylamide concentrations below that of gelation and in the presence of 0.5 % agarose, while the plots of the corresponding free 155 bp DNA fragments are linear in that range. The curvature presumably reflects the size increase concomitant with complex formation, since DNA larger than 155 bp also exhibits that curvature on polyacrylamide stabilized by nonrestrictive agarose gels.