bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of acrylamido‐buffers for isoelectric focusing by capillary zone electrophoresis
Authors: Righetti Pier Giorgio , Ettori Claudia , Chiari Marcella
Published: May 2005

Immobilized pH gradients use a series of weak acrylamido acids and bases (Immobiline) to create a pH gradient along the separation axis. These buffers can be degraded in water by two mechanisms: (i) hydrolysis of the amido bond, with generation of free acrylic acid and either an amino acid or a diamine; (ii) autopolymerization to oligomers and/or n‐mers. In order to check for these degradation products, different capillary zone electrophoresis systems for analysis of all Immobilines have been devised. The acidic compounds are resolved in 100 mM acetate, pH 4.0, whereas the alkaline Immobilines are separated in 50 mM phosphate buffer, pH 7.7 (or pH 7.2 for the weaker species). Polymers of alkaline Immobilines are resolved in 50 mM phosphate buffer, pH 2.5, in 1 % Ficoll‐400. All Immobilines are detected underivatized, by their adsorption at 214 or 254 nm. A calibration curve has been constructed for quantification of acrylic acid contamination. As little as 1 mol% of acrylic acid contamination in Immobiline solutions can be detected, with a sensitivity limit below 0.2 mM (at the injection port).
Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 8‐aminonaphthalene‐1,3,6‐trisulphonic acid: Application to the enzymological structural analysis of oligosaccharides
Authors: Jackson Peter , Williams Gwynn R.
Published: May 2005

Mono‐ and oligosaccharides, each containing a reducing end group, were labeled with the charged fluorophore 8‐aminonaphthalene‐1,3,6‐trisulphonic acid and the resulting derivatives were separated with high reselution by polyacrylamide gel electrophoresis using a method developed recently (P. Jackson, Biochem. J. 1990, 270, 705–713) but with an alternative electrophoretic buffer system. The fluorescent derivatives of glucose and all its straight chain, α 1–4 linked, oligomers from maltose to maltoheptaose were well resolved. Various isomers such as maltose and lactose could be separated, as were maltose and cellobiose and some epimers, for instance glucose and galactose. The method was applied to the analysis of the partial sequential degradation of a complex oligosaccharide with neuraminidase and β‐galactosidase. Gels showing fluorescent saccharide band patterns were recorded in picomolar quantities either photographically or using an imaging system based on a cooled charge‐coupled device.
Searching for genetic markers for hereditary diseases in cattle by means of DNA fingerprinting
Authors: Doff Gaudenz , Glowatzki Marie‐Louise , Gaillard Claude
Published: May 2005

Losses caused by inherited diseases affect not only the breeder's income but also the breeding programs themselves. In Switzerland the cardiomyopathy of cattle and the syndrome of arachnomelia and arthrogryposis are currently being investigated in order to find genetic markers for these diseases. It seems that DNA fingerprinting with multilocus probes is the most efficient approach available at present. Microsatellites analyzed with the polymerase chain reaction technique will provide a usable, marker map within the next ten years and will therefore provide a perfect tool to find markers for hereditary diseases.
Hybridization and polymerase chain reaction amplification of simple repeated DNA sequences for the analysis of forensic stains
Authors: Roewer Lutz , Rieß Olaf , Prokop Otto
Published: May 2005

We have evaluated oligonucleotide hybridization and amplification techniques with regard to quantity and quality of genomic DNA that is under investigation in practical forensic case work. In order to obtain sufficient information from analyzing stain material, we use hypervariable simple repeat sequences for individualization, which occur in all eukaryotic genomes. For the analysis of larger amounts of stains (> 500 ng DNA) the multilocus probes (CAC)5/(GTG)5

Probes are written in capitals, genomic noncoding sequences in lower case

are superior because of their discrimination potential – provided that the hybridizing DNA is of high molecular weight. The less discriminating probes (CT)8 and (GACA)4 are more sensitive (minimal amount: 100ng DNA) and still informative when the DNA is degraded. To increase the sensitivity of forensic stain analysis in special cases we have used the polymerase chain reaction technique to amplify hypervariable simple (gt)n/(ga)m repeat structures from the intron 2 of HLA‐DRB genes. Largely independent of the starting amount of DNA and independent of the degradation status, we were able to generate discriminating DNA fragments, which can be used to type (i) microstains and (ii) totally degraded material including human mummy DNA.
Chromosomal organization of simple repeated DNA sequences
Authors: Nanda Indrajit , Zischler Hans , Epplen Conny , Guttenbach Martina , Schmid Michael
Published: May 2005

Stretches of short, simple DNA sequences are widespread in all eukaryote genomes studied so far. Simple sequences are thought to undergo frequent expansion and deletion due to intrinsic genomic mechanisms. Some of the simple sequences were used successfully to detect hypervariable loci in various genomes. Hybridization experiments using synthetic probes not only revealed the informative simple repeats suitable for DNA fingerprinting in a particular specie, but also reflected the wide range of distribution of the simple sequences among eukaryotes. The organization of these simple repetitive sequences at the chromosomal loci was investigated using in situ hybridization with chemically synthesized, pure oligonucleotide probes. Both biotin‐ and digoxigenin‐attached probes detected specific chromosomal sites that are enriched in the respective simple‐repeat blocks. Depending on the organism and probe used, accumulation of simple DNA sequences at individual or multiple sites on the chromosomes of different vertebrates could be demonstrated. The simple repetitive DNA sequences are located in different chromosomal regions (e. g., heterochromatin on the sex chromosomes, nucleolus organizer regions, and R‐band sites), which are constrained considerably during evolution.
Two‐dimensional electrophoresis in small gels for applications in veterinary medicine
Authors: Miller Ingrid , Gemeiner Manfred
Published: May 2005

A versatile method for two‐dimensional electrophoresis that can be performed easily, even in small routine laboratories, is described. The procedure combines a first‐dimensional isoelectric focusing run in a PhastSystem with denaturing electrophoresis in a small vertical electrophoresis chamber. The described arrangement of two first‐dimensional gel strips in the second dimension allows direct comparison of two related samples, eliminating most of the artifacts that usually lead to misinterpretations. The presented procedure can be conveniently adapted to the needs of each laboratory; at present, it is being extensively used to establish proteir patterns of healthy individuals of different species, a prerequisite with regard to help and support diagnosis.
Efficient, small scale electroelution of high molecular weight DNA from agarose gels by a miniature vertical electrophoresis cell
Authors: Pascali Vincenzo L. , Pescarmona Marina , Dobosz Marina , d'Aloja Ernesto
Published: May 2005

A miniature, vertical electrophoretic system is described that allows quick elution and efficient harvesting of double‐stranded, high molecular weight DNA fragments from agarose gels. A gel slice containing the band of interest is excised from the agarose gel after submarine electrophoresis and placed into an electrophoresis cell, assembled by inserting a truncated micropipette tip into a 2.0 mL polypropylene tube. The tip supports the agarose slice and is connected to dialysis tubing. Platinum wire electrodes are placed over the tip and at the bottom of the test tube. DNA molecules are quickly eluted into the dialysis bag (60 s to 10 min for bands ranging from 2 to 23 kb) and then easily recovered. The method is highly efficient and allows considerable time saving. Of special interest is its applicability to small starting amounts of DNA (<0.5 μg per band). Purity of the DNA is comparable, if higher, to that obtainable by conventional electroelution on dialysis bags and DEAE–cellulose membrane electrophoresis. The system is simple enough to be used as a routine method for eluting multiple bands in short times.
A new multiphasic buffer system for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100 000–1000, and their detection with picomolar sensitivity
Authors: Wiltfang Jens , Arold Norbert , Neuhoff Volker
Published: May 2005

A novel multiphasic buffer system for high resolution sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100 000–1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis [1–3], allows complete stacking and destacking of proteins/ peptides within the above Mr, range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counterions – the characteristic feature of the present ionic system – the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate‐proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr, 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion‐exchange of polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloi dal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the bufferion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively.
High‐performance capillary electrophoresis of gangliosides
Authors: Liu Yuanbin , Chan K.‐F. Jesse
Published: May 2005

Gangliosides are sialic acid‐containing glycosphingolipids. In aqueous media, these glycolipids have been shown to exist as stable micelles. Ganglioside micelles could be analyzed by high‐performance zonal capillary electrophoresis in uncoated fused‐silica capillaries within 10 min. The mass sensitivity determined by monitoring the absorption of ultraviolet light at 195 nm was in the order of 10−11 mol. Increasing the pH of the running buffer from 3.0 to 7.4 or the voltage from 10 to 30 kV increased the relative mobilities of gangliosides. By contrast, increasing the ionic strength of the buffer decreased the migration and broadened the elution peak widths of gangliosides. Ganglioside

The abbreviations used for gangliosides are according to the recommendations of the Nomenclature Committee, International Union of Pure and Applied Chemistry [46]. The basic designation of ganglio‐series gangliosides are based on the Svennerholm system [47]. For example: GMl, II3NeuAc‐GgOse4Cer.

micelles including GM1, GD1b and GT1b were resolved into separate peaks by capillary electrophoresis at physiological pH shortly after mixing. Upon prolonged inc ubation, the ganglioside peaks merged to form a single species. The fusion process was temperature‐dependent. At 50°C, formation of mixed micelles between polysialogangliosides GD1b and GT1b was complete within 30 min. In contrast, no fusion of the ganglioside peaks was observed at 0°C even after 75h. Formation of mixed micelles between GD1b and other polysialogangliosides including GD1a, GT1b and GQ1b at 37°C required 1.5, 3.0 and 2.0 h, respectively. Formation of mixed micelles between monosialoganglioside GM1 and polysialogangliosides were 6‐ to 36‐fold slower. No fusion was observed between monosialogangliosides GM1 and GM2 after 2 days of incubation. These findings indicate that polysialogangliosides may have higher propensities than monosialoganglioside to form mixed micelles.
Quantitative analysis of two‐dimensional protein profiles of inbred lines of maize (Zea mays L.)
Authors: Higginbotham Jeri Welsh , Smith J. Stephen C. , Smith Oscar S.
Published: May 2005

Two‐dimensional electrophoresis and fluorography of [35S] methionine labeled maize germinated embryo proteins were performed at Cold Spring Harbor Laboratory. Fluorographs of 63 gels representing 37 inbred lines were subsequently scanned and spot‐detected at Protein and DNA Imageware Systems (Huntington Station, NY). The digitized images were then matched with the aid of PDQUESTII computer software. Over 1500 different protein spots were included in the resulting dataset. The optical density data were normalized to parts per million, then transformed to their natural logarithms. Analyses of variance were performed on each spot in order to select for further study those spots with most of their variation partitioned among inbred lines rather than within inbred lines. Using this method of spot selection, over 100 protein spots were included in the set of spots which display significant differences among inbred lines of maize.
The simplified technique of high resolution two‐dimensional polyacrylamide gel electrophoresis: Biomedical applications in health and disease
Authors: Marshall Thomas , Williams Katherine M.
Published: May 2005

The application of our simplified technique of high resolution two‐dimenional polyacrylamide gel electrophoresis (2‐D PAGE) to human body fluids is reviewed. Serum/plasma protein changes associated with alcohol abuse, familial dyslipoproteinemia (“fish‐eye” disease), and myocardial infarction are demonstrated. High resolution 2‐D PAGE of amniotic fluid, cerebrospinal fluid, urine, and saliva is shown with reference to the work of others, and the detection of pink‐violet staining “lumicarmines” in sweat and tear fluid is reported for the first time. General aspects relating to the methodology are discussed. These include sample preparation, the choice of electrophoresis conditions (denaturing or nondenaturing) and detection method (Coomassie Brilliant Blue or silver), and the effects of native protein pretreatment with sodium dodecyl sulfate prior to silver staining or isoelectric focusing gel shrinkage in glycerol prior to second‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.
Use of a fast protein electrophoretic purification procedure for N‐terminal sequence analysis to identify S‐locus related proteins in stigmas of Brassica oleracea
Authors: Gaude Thierry , Denoroy Luc , Dumas Christian
Published: May 2005

In the cruciferous plant Brassica oleracea L. (cabbage), the S‐locus specific glycoproteins (SLSGs) isolated only in stigmas are considered to play an important role in the normal prevention of self‐fertilization. Recent molecular data have shown that the gene encoding these glycoproteins (the SLG gene) belonged to a multigenic family consisting of about 10 homologous copies among which an, other member is expressed, the S‐locus related gene (SLR1gene). Our aim was to determine whether the SLR1‐gene proteins were expressed in the stigmatic tissues. We first identified the putative SLSGs or SLR1‐proteins by Con A‐peroxidase detection of glycoproteins separated after isoelectric focusing in polyacrylamide gels. We describe a fast purification procedure for the glycoproteins of interest, based on analytical isoelectric focusing, electrophoresis, and electroblotting of proteins onto polyvinylidene difluoride membranes. Blotted proteins were sequenced for N‐terminal amino acid determination. By comparison of the N‐terminal sequences of the purified proteins with the peptide sequence predicted from the SLR1‐cDNA, we demonstrate the expression of SLR1‐like proteins in stigmas of B. oleracea.
Genetic polymorphisms of the A and B subunits of human coagulation factor XIII in mainland Italy and Sardinia: Description of a new FXIIIA variant allele
Authors: Scacchi Renato , Corbo Rosa Maria , Mulas Giuseppe , Mureddu Libero , Pascone Roberto
Published: May 2005

The distribution of the two alleles of FXIIIA and the three alleles of FXIIIB were studied in populations from mainland Italy and from Sardinia. The frequencies of the FXIIIA*2 allele were 0.266 and 0.265. The frequencies of FXIIIB*1 were 0.787 and 0.765; of B*2, 0.070 and 0.094; of B*3, 0.143 and 0.141. A new cathodal FXIIIA allele (A*7) was described in the Rome sample. No significant difference in the distribution of allele frequencies for either system was found between the two populations studied. For typing both markers, good results were also obtained by using whole blood instead of plasma.
The characterization and use of different antibodies against the hsp70 major heat shock protein family for the development of an immunoassay
Authors: Margulis Boris A. , Nacharov Peter V. , Tsvetkova Olga I. , Welsh Michael , Kinev Alexander V.
Published: May 2005

The hsp70 family of major stress proteins is composed of several different members exhibiting similar structural and functional properties. In order to obtain an antiserum with wide epitope reactivity, rabbits were immunized with a mixture of native and denatured hsp70 purified from bovine muscle by ATP‐affinity chromatography. Screening for antibody specificity was performed by a “sandwich” enzyme linked immunosorbent assay (ELISA). Immunoprecipitation and immunoblotting analyses demonstrated that the polyclonal antiserum obtained by us and a monoclonal antibody raised against a different preparation of antigen recognized the same determinant on the native hsp70 molecule (inducible form). With a different specificity the polyclonal antiserum recognized only the denatured monomers of the other members of the hsp70 family. These results are discussed in relation to the immunological features of the hsp70 molecule and to the development of an immunoassay for the detection of hsp70 in Cell and tissue extracts.
Improved method for identification of proteins using two‐dimensional electrophoresis with immobilized pH gradient isoelectric focusing
Authors: Mackin Robert B. , Raker Veronica A. , Spiess Joachim
Published: May 2005

Recent advances in protein sequence analysis now permit the determination of partial N‐terminal and internal primary structure from low picomole quantities of protein. The major remaining hurdles to sequence analysis of small amounts of protein are the identification, isolation, and handling of microgram and submicrogram quantities of protein. The technique of two‐dimensional Electrophoresis using immobilized pH gradient isoelectric focusing circumvents many of these problems. However, poor correlation between the first and second dimension have prevented use of this technique for the identification of some proteins which can only be assayed prior to the denaturing conditions used in the second dimensional sodium dodecyl sulfate‐polyacrylamide gel Electrophoresis procedure. An improved method is presented which allows correlation of the native biological activity (first dimension) to a silver stained protein (second dimension) with a high degree of confidence.
The MELANIE project: From a biopsy to automatic protein map interpretation by computer
Authors: Appel Ron D. , Hochstrasser Denis F. , Funk Matthieu , Vargas J. Reynaldo , Pellegrini Christian , Muller Alex F. , Scherrer Jean‐Raoul
Published: May 2005

The goals of the MELANIE project are to determine if disease‐associated patterns can be detected in high resolution two‐dimensional polyacrylamide gel electrophoresis (HR 2D‐PAGE) images and if a diagnosis can be established automatically by computer. The ELSIE/MELANIE system is a set of computer programs which automatically detect, quantify, and compare protein spots shown on HR 2D‐PAGE images. Classification programs help the physician to find disease‐associated patterns from a given set of two‐dimensional gel electrophoresis images and to form diagnostic rules. Prototype expert systems that use these rules to establish a diagnosis from new HR 2D‐PAGE images have been developed. They successfully diagnosed cirrhosis of the liver and were able to distinguish a variety of cancer types from biopsies known to be cancerous.
A two‐dimensional electrophoresis‐related laboratory information processing system: Spot matching
Authors: Kuick Rork D. , Skolnick Michael M. , Hanash Samir M. , Neel James V.
Published: May 2005

An approach for the computer‐assisted analysis of two‐dimensional gels has been developed as a part of our laboratory information processing system (LIPS). This approach relies in part on an algorithm for the pairwise matching of protein spots. The matching process initially matches spots based on a cross‐correlational measure of how well neighboring spots align. While this first pass correctly determines most spot correspondences and noncorrespondences, it can make errors. Higher accuracy is obtained by monitoring the consistency of spot match decisions in a second pass, which demands that neighboring spot pairs that align spatially must also have been found to match in the first pass. Pairwise comparisons of gels are, combined into n‐way comparisons by matching spot lists of gels to “master” gel spot lists, which in turn are matched to higher level masters, resulting in a hierarchy of matched spots. After each pairwise match the results are reviewed and corrected with the assistance of a graphical match‐editor. Results are given for 19, single‐Cell‐derived lymphoid clones in which the presence of a mutation had previously been established, each processed in duplicate. Only one of 46 spot changes failed to be detected, which demonstrates that the strategy is sensitive and efficient for detecting qualitative spot differences.
Immobilized pH gradients (IPG) simulator ‐ an additional step in pH gradient engineering: II. Nonlinear pH gradients
Authors: Righetti Pier Giorgio , Tonani Carlo
Published: May 2005

While in the companion paper (Tonani, C. & Righetti, P. G., Electrophoresis 1991, 12, 1011–1021) we gave the general outline of our new computer program, immobilized pH gradients (IPG) simulator, able to simulate and optimize linear pH gradients for isoelectric focusing in immobilized pH gradients, in the present report we extend the application of such a program to: (i) convex exponential gradients, (ii) logarithmic and (iii) polynomial gradients

Program available from Fluka Chemie AG, Buchs, Switzerland

. Such gradients are meant to give equal space to protein spots in complex protein mixtures (e.g., cell lysates, biological fluids) and follow the statistical distribution of protein pI values along the pH axis. They will prove of fundamental importance in two‐dimensional maps, both because they optimize the spreading of spots in the two‐dimensional plane and because of the excellent reproducibility of immobilized pH gradients. The following concave exponential recipes are given: pH 3–8, pH 3–9, pH 3–10, pH 3–11, pH 4–7, pH 4–8, pH 4–9, pH 4–10, pH 4–11, pH 5–8, pH 5–9, and pH 5–10, as well as the most extended pH 2.5–11 interval. Two interesting logarithmic gradients are described: pH 3–6 and pH 3–7 and one sigmoidal (derived with a polynomial of 5th degree): pH 3–11.
Capillary electrophoresis of DNA in agarose solutions at 40°C
Authors: Boček Petr , Chrambach Andreas
Published: May 2005

DNA fragments ranging from 72 to 1353 by in length (ϕX174 RF DNA/HaeIII) were separated by capillary electrophoresis in 0.3–2.0% solutions of agarose (SeaPlaque GTG) at 40°C. Liquified agarose above its gelling temperature is easily filled and refilled into capillaries. Its background absorbance at 260 nm was sufficiently low to allow for DNA detection at an estimated DNA load of 13 ng/10 components. Sample injection proceeded at 1 kV for 16s. The internal capillary diameter was 150 μ, the migration path 27 cm. Migration times varied from 5 to 14 min at 185 V/cm. Potentially, the applicability of capillary electrophoresis in agarose solutions can be expected to extend to the entire size range of DNA, in view of the recent demonstration of kb‐sized circular DNA separations in agarose solutions, and those of Mb‐sized DNA‐agarose complexes in linear polyacrylamide solutions.
Capillary electrophoresis in agarose solutions: Extension of size separations to DNA of 12 kb in length
Authors: Boček Petr , Chrambach Andreas
Published: May 2005

The upper limit of the size range of DNA amenable to separation in agarose solutions above their gelling temperature, using capillary zone electrophoresis apparatus, was increased to 12 kb. The plot of log(bp)vs. mobility derived from electrophoresis in 1.7% agarose solution is biphasic, exhibiting higher resolving power for DNA less than 1 kb in size than that of larger sizes. Resolving power for DNA larger than 1 kb increased when the agarose concentration was increased in the range of 1.0–2.6%. It was similar in solutions at 40°C of SeaPrep and SeaPlaque agaroses as well as in Acrylaide (trade names are those of the manufacturer). However, the resolving power of SeaPrep agarose at 25°C was inferior to that at 40°C. Concave plots of log(mobility) vs. concentration of the agarose solutions are those predicted under the assumption that the effective “equivalent radius” of the DNA molecule diminishes with increasing agarose concentration in the investigated concentration range up to 2.6%.
Continuous, on‐line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus
Authors: Middendorf Lyle R. , Bruce John C. , Bruce Robert C. , Eckles Robert D. , Grone Daniel L. , Roemer Stephen C. , Sloniker Gary D. , Steffens David L. , Sutter Scott L. , Brumbaugh John A. , Patonay Gabor
Published: May 2005

A new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state‐of‐the‐art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with > 99% accuracy.
High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels
Authors: Mozdzanowski Jacek , Hembach Peter , Speicher David W.
Published: May 2005

Optimal conditions of electroblotting that led to high protein recovery on polyvinylidene difluoride (PVDF) membranes were determined for sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). SDS concentrations in the gel and transfer buffer were found to be the most important factors affecting the amount of protein recovered on the PVDF membrane. The largest loss occurred during the first 10–30 min of transfer due to the relatively high initial SDS concentration in the gel. During this initial stage of transfer, most of the protein passed through the primary membrane and was partially retained on secondary and tertiary membranes. The value of presoaking gels prior to transfer to reduce the amount of SDS was evaluated by quantitating free SDS densitometrically and by correlating the reduced SDS concentration with increased electroblotting efficiency from presoaked gels. Transfer time was evaluated and no “overtransfer” was found even after very long transfer times. These results clearly indicate that proteins electroblotted onto PVDF membranes were tightly bound and could not be released by extending the transfer time. The effects of methanol and SDS concentrations on protein adsorption from solution to PVDF were also determined quantitatively. The results of this study strongly suggest that proteins fully saturated with SDS cannot bind efficiently to PVDF membranes. Since SDS is necessary for high protein mobility, the challenge in efficient electroblotting is to maintain an optimal SDS concentration which is high enough to permit effective removal from the gel and low enough to permit effective binding to the PVDF membrane. For 1.5 mm thick gels containing 0.2% SDS, presoaking the gel for 15–20 min in transfer buffer with 10% methanol prior to electroblotting provided the best recovery on the primary membrane.
Temperature‐gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction
Authors: Riesner Detlev , Steger Gerhard , Wiese Ulrich , Wulfert Michael , Heibey Monika , Henco Karsten
Published: May 2005

In order to detect mutations in a gene, either known mutations from human diseases or artifical ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarentees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo‐ and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of <15 %. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.
Nonradioactive ribonuclease protection analysis using digoxygenine labeling and chemiluminescent detection
Authors: Wundrack Iris , Dooley Steven
Published: May 2005

A sensitive nonradioactive ribunouclease protection assay is described which we have used to study c‐myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with amplified gene. For in vitro transcription we used digoxygenine (DIG)‐rUTP instead of [α‐32P]CTP or [α‐32P]UTP and after polyacrylamide gel electrophoresis the protected probes were transferred to a nylon membrane from Boehringer Mannheim using electroblotting. Subsequently the membrane was analyzed by chemiluminescent detection. Results were obtained after 1 h of exposure and were comparable with those using radioactivity.
Native horizontal ultrathin polyacrylamide gel electrophoresis of proteins under basic and acidic conditions
Authors: Heukeshoven Jochen , Dernick Rudolf
Published: May 2005

The 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

The 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

Mouse 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

This 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

N‐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

High 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

Vascular 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

The 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

Quantitative 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

An 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

The 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

This 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

The 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

Human 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

Two‐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).