bluuebunny/crossref_metadata_2025_split huggingface dataset:
Native horizontal ultrathin polyacrylamide gel electrophoresis of proteins under basic and acidic conditions
Authors: Heukeshoven Jochen , Dernick Rudolf 
Published: May 2005

AbstractThe preparation of homogeneous ultrathin native polyacrylamide gels, using a basic as well as an acidic buffer system is described. The basic buffer system consists of Tris‐HC1/Tris‐glycine, the same buffer as in sodium dodecyl sulfate (SDS)‐gel electrophoresis but without SDS. The acidic system uses potassium acetate, pH 4.3, as gel buffer and β‐alanine, pH 4.6, acetic acid as electrolytes. The gels are covalently bound on glass plates. Binding of acidic gels requires a special pretreatment of glass plates. The whole procedure is simple and extraordinarily fast: 100–120 min from the start of gel preparation to the end of electrophoresis. Coomassie staining is done in 40 min and silver staining in 90 min. The native gels are excellently suited for diffusion blotting. Further attractive properties of these gels are easy handling, simple drying and dimensional stability.
Mass spectrometric analysis of blotted proteins after gel electrophoretic separation by matrix‐assisted laser desorption/ionization
Authors: Eckerskorn Christoph , Strupat Kerstin , Karas Michael , Hillenkamp Franz , Lottspeich Friedrich 
Published: May 2005

AbstractThe molecular masses of electroblotted proteins were determined with a time‐of‐flight mass spectrometer by matrix‐assisted laser desorption/ionization directly from blot membranes. Therefore standard proteins, separated by polyacrylamide gel electrophoresis, were electroblotted onto polyvinylidene difluoride or polyamide membranes by standard procedures. Pieces of membrane containing the protein of interest were soaked in matrix solution and analyzed in the mass spectrometer.
Protein synthesis in murine organs during postimplantation development detected by two‐dimensional gel electrophoresis
Authors: Praxmayer Claudia , Murach Karl‐Friedrich , Baumgartner Bernhard , Aberger Friedrich , Schlegel Elisabeth , Illmensee Karl 
Published: May 2005

AbstractMouse embryos were isolated from the uterus on days 10 to 11 of gestation and incubated in Dulbecco's modified Eagle's medium (DMEM) with [35S]methionine for 4 h. Subsequently, their hearts and the brains were dissected. The brain was divided into three parts, containing the telencephalon, mesencephalon, and myelencephalon. These tissues were then processed for two‐dimensional (2‐D) gel electrophoresis. Protein synthesis of the isolated tissues was analyzed for organ‐and cell lineage‐specific patterns. We studied proteins with isoelectric points (pI) ranging from 4 to 10 and relative molecular weights (Mr) varying from 10000 to 200000 and found several significant quantitative and qualitative differences between the tissues and the developmental stages analyzed. In particular, we were able to distinguish between protein spots that we now attribute putatively to the corresponding embryonic organs. These differences may reflect some of the organ‐ and cell lineage‐specific changes in protein synthesis and gene expression during early mammalian differentiation.
A human myocardial two‐dimensional electrophoresis database: Protein characterisation by microsequencing and immunoblotting
Authors: Baker Cathy S. , Corbett Joseph M. , May Andrew J. , Yacoub Magdi H. , Dunn Michael J. 
Published: May 2005

AbstractThis communication briefly describes how a human heart two‐dimensional electrophoresis (2‐DE) protein database is being established in our laboratory. The database contains more than 1500 polypeptides and approximately fifty proteins from 2‐DE gels of human myocardial tissue have been characterised. Information about the proteins has been compiled including molecular weight (Mr), isoelectric point (pI), sample spot (SSP) number, protein name, partial sequence, and antibody reacting with the protein. The first stage of this project involves the investigation of protein with pIs in the range pH 4–7. Future studies will employ immobilised pH gradient (IPG) gels as the first dimension of the 2‐DE to examine basic proteins. The ultimate goal of this project is to establish a global picture of human heart protein expression in both normal and disease conditions.
Identification of human myocard proteins separated by two‐dimensional electrophoresis
Authors: Jungblut Peter , Otto Albrecht , Regitz Vera , Fleck Eckart , Wittmann‐Liebold Brigitte 
Published: May 2005

AbstractN‐Terminal sequencing, internal sequencing and amino acid analysis were used to identify twelve proteins of the human myocard two‐dimensional gel electrophoresis (2‐DE) pattern. Amino acid analysis was shown to be a powerful tool in addition to sequencing. The identification of a disease‐associated N‐terminally blocked protein by internal sequencing was not successful. The twelve identified proteins are the basis of a human myocard 2‐DE database.
Application of high resolution two‐dimensional polyacrylamide gel electrophoresis of polypeptides from cultured neonatal rat cardiomyocytes: Regulation of protein synthesis by catecholamines
Authors: He Chang , Müller Ursula , Oberthür Walter , Werdan Karl 
Published: May 2005

AbstractHigh resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was applied to cultured neonatal rat heart muscle cells, incubated for 72 h at 37°C in serum‐free medium, either in the absence or in the presence of 0.1 μM norepinephrine. After silver staining, about 340 and 550 protein spots could be seen in cardiomyocytes, cultured either in the absence or presence of norepinephrine. Of these spots, 141 could be further characterized according to isoelectric point and molecular weight, with 71 protein spots being present under both conditions. In cells cultivated in presence of norepinephrine, 58 new protein spots appeared, whereas 12 spots disappeared, and 22 spots increased (whereas 3 spots decreased) in intensity. In comparison with 2‐D PAGE of rat cardiomyocytes, the protein pattern of the intact heart of neonatal rats is incongruent. 2‐D PAGE of polypeptides of cultured neonatal rat cardiomyocytes may be a suitable tool to study the regulation of protein synthesis by various stimuli with relevance to cardiac growth adaptation, inotropy and heart failure.
Comparison of the cellular protein composition of aortic smooth muscle cells in vivo and in vitro by two‐dimensional gel electrophoresis
Authors: Weiß Hans‐Dieter , Betz Eberhard , Karsch Karl Rüdiger 
Published: May 2005

AbstractVascular smooth muscle cells are of major interest for the evaluation of atherosclerotic processes. By the use of two‐dimensional gel electrophoresis and subsequent silver staining we investigated the protein expression of smooth muscle cells in the native thoracic aorta, immediately after enzymatic disaggregation, in subconfluent proliferating and in postconfluent nonproliferating primary cell cultures. Compared to the native thoracic aorta the protein composition of smooth muscle cells in cell culture is changed dramatically. Furthermore, significant differences in protein expression between proliferating and nonproliferating smooth muscle cells in cell culture were found.
Genetic polymorphism of apolipoprotein H (β2‐glycoprotein I) in African blacks from the Ivory Coast
Authors: Cleve Hartwig , Vogt Ulrike , Ilyas Kamboh M. 
Published: May 2005

AbstractThe apolipoprotein H (APO H) polymorphism was analyzed in the Negroid population from the Ivory Coast using polyacrylamide gel isoelectric focusing, followed by immunoblotting. The gene frequencies of alleles APO H*1, APO H*2, APO H*3 and APO H*4 were calculated to be 0.012, 0.921, 0.047 and 0.020, respectively. The assumption that APO H*4 represents a Negroid marker allele is supported by this population study.
Quantitative two‐dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes
Authors: Miller Mark J. , Maher Veronica M. , Justin McCormick J. 
Published: May 2005

AbstractQuantitative two‐dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin‐derived human fibroblast cell line (LG 1) and three immortal derivatives of LG1. One derivative, designated MSU–1.1 VO, was selected for its ability to grow in the absence of serum and is non‐tumorigenic in athymic mice. The other two strains were selected for focus‐formation following transfection with either Ha‐ras or N‐ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two‐dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU–1.1 VO, and the ras transformed cell strains. The MSU‐1.1 VO cells were more closely related to the parental LG1 than to the ras‐transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras‐transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha‐ras and N‐ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha‐ras transformed cells than the N‐ras. These results demonstrate the potential of automated two‐dimensional gel analysis for classifying different gel patterns, even patterns whose differences are obscured by the minor changes in spot intensity that arise between separate cell cultures.
The terminator, an apparatus for simultaneous homogenization of 96 small seeds individually
Authors: van den Berg Bartel M. , Tamboer Jan H. A. 
Published: May 2005

AbstractAn apparatus was developed to homogenize 96 individual seeds of tomato, pepper, cabbages, and other seeds of similar size in a 96‐well tissue culture plate in 45 s. The apparatus, named the terminator, consists of two parts: a base plate that can hold the 96‐well tissue culture plate, and a vibrating part that is attached to a variable transformer. The seeds are homogenized by the vibrating action of 96 small homogenizers that are attached to the vibrating part and that fit perfectly into the wells of the tissue culture plate. The seed homogenates made in this way can be used for electrophoretic analysis of genetic variations of seed proteins.
Recovery of protein by Coomassie Brilliant Blue precipitation prior to electrophoresis
Authors: Marshall Thomas , Williams Katherine M. 
Published: May 2005

AbstractThe interaction of protein with Coomassie Brilliant Blue G‐250 results in formation of an insoluble protein‐dye complex which can be recovered by centrifugation and redissolved for electrophoretic analysis. The precipitated protein can be washed in acetone to remove excess dye in order to enhance resolution. The residual dye becomes dissociated from the proteins on electrophoresis and can be exploited as a “dye front”. The method allows simultaneous protein assay and recovery of microgram amounts of protein from dilute solution and could be widely applied for conserving, concentrating and desalting minute amounts of valuable sample prior to electrophoretic analysis.
Human liver protein map: A reference database established by microsequencing and gel comparison
Authors: Hochstrasser Denis F. , Frutiger Séverine , Paquet Nicole , Bairoch Amos , Ravier Florence , Pasquali Christian , Sanchez Jean‐Charles , Tissot Jean‐Daniel , Bjellqvist Bengt , Vargas Reynaldo , Appel Ron D. , Hughes Graham J. 
Published: May 2005

AbstractThis publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two‐dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.
The relative importance of transport phenomena in recycling isoelectric focusing
Authors: Burgaud Caroline , Clifton Michael J. , Sanchez Victor 
Published: May 2005

AbstractThe various transport phenomena involved in recycling isoelectric focusing are analyzed for their contributions to band spreading so as to find ways of improving the resolution of this liquid‐phase protein purification method. A numerical model is proposed that takes into account diffusion, electroosmosis and electrophoretic migration as a function of pH. The electrohydrodynamic effects have so far been neglected in these calculations. The results of these calculations are compared with experimental measurements performed in different chamber geometries, with a variety of proteins and under different operating conditions, always chosen to avoid flow instabilities. This comparison shows that the resolution of this process is greatly impaired if the electroosmotic slip velocity at the wall is not suppressed.
Electrophoretic study of conformational changes of a human soluble β‐D‐galactoside‐binding lectin upon storage
Authors: Avellana‐Adalid Virginia , Joubert‐Caron Raymonde , Caron Michel , Bladier Dominique 
Published: May 2005

AbstractHuman brain lectin (HBL), a β‐galactoside specific soluble lectin, was purified by affinity chromatography. An alkylated derivative of this lectin was also prepared. Both native and modified molecules were conserved at − 20°C in the presence or absence of β‐mercaptoethanol, a reducing agent which was described to maintain the lectin activity in vitro or in the presence of β‐mercaptoethanol and lactose. The impact of storage conditions, over one year, on the native and derivated lectins, was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, isoelectric focusing and titration curve, using the PhastSystem (Pharmacia). Western‐blot analysis using an anti‐HBL antibody and size‐exclusion high performance liquid chromatography were used to complete the study. The subunit Mrs were estimated before freezing (T0) and after three and twelve months (T3, T12). They were comparable for all preparations. In all samples tested, isoelectric focusing demonstrated the existence of at least three acidic proteins, with the pI ranging between 4.7–4.9. Titration curves clearly showed pH‐dependent conformational changes, resulting in a panel of differently charged molecular species, some of which may be related to different oxidative states of the cysteine residues. We concluded that lectin can be stored at –20°C for at least one year before use as a reagent since the modifications revealed by electrophoretic analysis do not alter the hemagglutination activity and carbohydrate binding properties. The immunoreactivity also remained unchanged.
Two‐dimensional electrophoretic analysis of immunoglobulin patterns in monoclonal gammopathies
Authors: Goldfarb Marcia F. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) electrophoresis was performed on 24 serum samples from patients diagnosed with monoclonal gammopathies. These samples had been shown to have a homogeneous immunoglobulin (M component) by zone electrophoresis and immunofixation. Using 2‐D electrophoresis, the nature of this aberrant protein was further analyzed. It has been presumed that the sharp, dark stained band identified by immunofixation was the production of a monoclonal immunoglobulin. The increased resolution afforded by 2‐D methodology reveals several different patterns. On 2‐D electrophoresis, a monoclonal antibody has a unique pattern. It consists of 3 to 6 strong, restricted heavy chain bands and a single distorted light chain spot. The 3–6 bands are microheterogeneity of the isoelectric point, attributed to posttranslational glycosylation and/or amidation/ deamidation. Analysis by 2‐D electrophoresis indicated only 5 samples with a true monoclonal pattern. All but 2 of the samples clearly had aberrant immunoglobulin, but interpretation of the pattern would suggest the protein is other than a fully synthesized monoclonal antibody. The samples showed the following: a monoclonal heavy chain pattern with multiple distorted light chain spots, only an aberrant light chain area, only an aberrant heavy chain, and only a polyclonal increase. Several IgG gammopathies had, in addition, concentrations of gamma heavy chain at a reduced size (34 kDa).
Selective influencing of the migration time of SO42− by forming neutral species with Pb2+ in capillary zone electrophoresis
Authors: Groh Thomas , Bächmann Knut 
Published: May 2005

AbstractOrganic and inorganic anions are analyzed using capillary zone electrophoresis with indirect fluorescence detection. As electrolyte 2,5‐dihydroxybenzoic acid is used. By adding Pb2+ to the elctrolyte the migration time of SO42− can be selectively influenced and the separation of C1− and SO42− considerably improved. The fluorescence intensity of 2,5‐dihydroxybenzoic acid increases in the presence of Pb2+ up to a concentration of 6*10−4 mol/L Pb2+. Limits of detection in the lower μmol/L range were obtained.
DNA digest analysis with capillary electrophoresis
Authors: Paulus Aran , Hüsken Dieter 
Published: May 2005

AbstractDNA digest analysis with polymer‐filled capillaries in capillary electrophoresis is described. The samples analyzed consisted of commercially available standards including a 100 base pair ladder with repeating units up to 2000 base pairs. Three DNA digests covered the most common small fragment ranges: up to 600 base pairs, up to 1500 base pairs and from 100 to 2500 base pairs. All samples were separated by traditional slab gel electrophoresis at various agarose concentrations and by automated capillary electrophoresis. The capillary electrophoretic separations were achieved with noncross‐linked polyacrylamide from 6% monomer solutions. The acrylamide was polymerized inside the capillary, which was coated with a methacryloxysilane to insure binding of the polyacrylamide to the capillary wall. With 6% columns, excellent separations were observed up to 600 base pairs with base line resolution for fragments differing in less than 10 base pairs. Resolution power for fragments between 600 and 2200 decreased to about 300 base pairs. Compared to slab gels, capillary electrophoresis achieved better resolution in the low fragment range, whereas with the reported column composition, slab gels were superior above 600 base pairs. Fast access to the analysis of a specific sample, automation, and a larger dynamic range for sample load are further benefits of a DNA digest analysis by capillary electrophoresis.
The silver staining procedure of sodium dodecyl sulfate‐gels may be accelerated by shortening fixation time
Authors: Kirkeby Svend , Moe Dennis , Bòg‐Hansen Thorkild C. 
Published: May 2005

AbstractIn most silver staining methods the first step in the staining of proteins separated after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis is a rather protracted fixation of the gels. Optimum fixation should be short, cause no background staining and effectively immobilize the proteins in the gel without masking the proteins for reaction with the staining solution. Further, the concentration of the fixing compounds should be as low as possible due to the potentional toxicity of fixatives. Fixation for only 5 min with mixtures of very low concentrations of formaldehyde and glutaraldehyde in ethanol, or a solution of formaldehyde or glutaraldehyde in picric acid and ethanol, fulfill these demands, provided that the gels were prefixed in ethanol‐acetic acid for 10 min. As a consequence of these results a fast and sensitive silver staining procedure is proposed.
Enhancing spot detection and reducing noise from digitized electrophoretic gel images using area processing filters
Authors: Patton Wayne F. , Tempst Paul 
Published: May 2005

AbstractTwenty area processing filters and filter combinations were evaluated in an effort to optimize presentation of two‐dimensional electrophoretic profiles to the Laplacian spot finder for maximal spot detection sensitivity. Images of electrophoresis gels were obtained by digitizing polyacrylamide gels at 1024 × 1024 picture elements (pixels) resolution with 256 grey scale levels using the charge coupled device (CCD) camera of the Millipore Bio Image 110S computerized imaging system. The images were imported into an Apple Macintosh microcomputer and selectively enhanced by applying various area processing filters. Previously described least squares fit, low‐pass, Gaussian and median filters were used to reduce noise in the digitized images. These filters differ in that during the summation process the least squares template weighs the immediately adjacent pixels more heavily than the Gaussian template. The low‐pass filters weigh all neighboring pixels equally. Median filters replace the pixel of interest with the middle (median) value of the pixel neighborhood. An analysis of convolution filter sizes indicated that a 7 × 7 matrix was optimal for 22 cm × 22 cm gels. When using the median area processing procedure, however, the 3 × 3 filter was found to be superior to the 7 × 7 filter. The 7 × 7 least squares filter significantly improved detection of low abundance polypeptides while having only minimal effects on the high abundance polypeptides. The 7 × 7 Gaussian and 3 × 3 median filters also improved detection of low abundance polypeptides but reduced the integrated areas of the high abundance polypeptides and thus their integrated optical densities as well. The 7 × 7 least squares and the 3 × 3 median filters effectively increased spot detection, and reduced noise with minimal alteration in the modal distribution of spot areas and integrated optical densities in the image. They also preserved image quality (minimal blurring). The practical consequence of using the 7 × 7 least squares, 3 × 3 median or 7 × 7 Gaussian filters is an 8‐fold increase in sensitivity due to noise reduction, resulting in the detection of 60–65% more polypeptides in a typical silver stained gel.
A new concept for separating nucleic acids by electrophoresis in solution using hybrid synthetic end labelled‐nucleic acid molecules
Authors: Noolandi Jaan 
Published: May 2005

AbstractAnalogy between the symmetry breaking of the electrical driving force and the opposing friction force in gels using pulsed electric fields is made with the corresponding effect for polyelectrolyte coils in solution related to the molecular weight‐independent charge density. The synthesis of hybrid molecules to break the symmetry of constant charge density is proposed, in which standard polypeptide end labels are attached to nucleic acid fragments. The combinatorial chemical library scheme of Brenner and Lerner (Proc. Natl. Acad. Sci. USA 1992, 89, 5381–5383), making use of hybrid molecules for another purpose, is mentioned, with the suggestion that for electrophoresis in solution a neutral end‐labelled polypeptide chain would give the largest molecular weight and time‐dependent change in the charge density for pulsed fields.
Detection by Nile Red of agarose gel electrophoresed native and modified low density lipoprotein
Authors: Greenspan Phillip , Gutman Robert L. 
Published: May 2005

AbstractThe use of Nile Red to track and stain low density lipoprotein (LDL) and modified LDL in agarose gels was investigated. Lipoproteins were prestained with Nile Red, a fluorescent dye, prior to electrophoresis. After 2 h of electrophoresis, the LDL and modified LDL were visualized using a UV transilluminator with an excitation wavelength of 302 nm. Spectrofluorometric analysis revealed that the Nile Red fluorescence of the stained LDL had an emission maximum of 609 nm. This rapid staining method of LDL and modified LDL can detect as little as 2.5 μg of LDL protein and permits the immediate visualization of these lipoproteins in agarose gels.