bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electrophoretic separation of multiprotein complexes from blood platelets and cell lines: Technique for the analysis of diseases with defects in oxidative phosphorylation
Authors: Schägger Hermann , Bentlage Herman , Ruitenbeek Wim , Pfeiffer Kathy , Rotter Stefan , Rother Christof , Böttcher‐Purkl Andrea , Lodemann Edgar 
Published: May 2005

AbstractA two‐dimensional electrophoretic technique combining blue native polyacrylamide gel electrophoresis (BN‐PAGE) with Tricine sodium dodecyl sulfate (SDS)‐PAGE was previously used for the localization of oxidative phosphorylation (OXPHOS) defects in human diseases starting from biopsy or autopsy tissues (Schägger, H., Electrophoresis 1995, 16, 763–770). In the present work the technique was extended for the resolution of OXPHOS enzymes from platelets and tissue‐cultured cells. Silver staining is required to detect the protein subunits of OXPHOS complexes in two‐dimensional gels. However, the use of cultured cells has major implications for patients with mitochondrial encephalomyopathies since it will reduce the number of invasive muscle biopsies. The ease of isolating the platelet membrane glycoprotein complex from a few milliliters of blood makes it possible to analyze this complex and its protein subunits in bleeding disorders like Glanzmann's thrombasthenia.
Novel acrylamido monomers with higher hydrophilicity and improved hydrolytic stability: II. Properties of N‐acryloylaminopropanol
Authors: Simò‐Alfonso Ernesto , Gelfi Cecilia , Sebastiano Roberto , Citterio Attilio , Righetti Pier Giorgio 
Published: May 2005

AbstractThe physico‐chemical properties and the electrophoretic behavior of the novel set of monomers reported by (Simò‐Alfonso et al. Electrophoresis 1996, 17, 723–731) have been evaluated. Of utmost importance was the combination of high hydrophilicity and extreme hydrolytic stability, most desired properties for any electrophoretic matrix, especially for protein fractionation. One of these monomers (N‐acryloylaminopropanol, AAP) was found indeed to be extremely hydrophilic (with a partition coefficient P of only 0.10, vs. P = 0.13 for N‐acryloylaminoethoxyethanol and P = 0.20 for acrylamide) and to possess excellent stability to alkaline hydrolysis. Its hydrolysis constant (0.008 L mol−1 min−1), as a free monomer, in an alkaline milieu, was found to be about one order of magnitude lower than conventional acrylamide (0.05 L mol−1 min−1). In the polymer state, the resistance to hydrolysis of poly(AAP) was assessed as 500 times greater than a conventional poly(acrylamide) matrix.
Comparative analysis of somatostatin analog peptides by capillary electrophoresis and micellar elektrokinetic chromatography
Authors: Idei Miklós , Mezö Imre , Vadász Zsolt , Horváth Anikó , Seprödi János , Érchegyi Judit , Teplán István , Kéri György 
Published: May 2005

AbstractCapillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC) methods, utilizing uncoated silica capillary and triethyl ammonium phosphate or sodium borate buffers in the pH range of 2.25–11.0, containing sodium dodecyl sulfate (SDS) (0–100 mM) for analysis of somatostatin‐analog peptides were developed. The method presented here was compared with the reversed‐phase high performance liquid chromatographic (RP‐HPLC) and CE methods developed for analysis of peptides. The peptides investigated in this work can be separated by CE on the basis of their electrophoretic mobility in aqueous buffer of low pH value (pH 2.25) or by MEKC on the basis of their hydrophobicity in SDS containing buffer of high pH value (pH 11.0). Optimal MEKC separation of the investigated peptides has been achieved at pH 11.0 in an Na‐borate buffer containig 100 mM SDS. CE at pH 2.25 proved insensitive to the hydrophobicity of the peptides investigated. By contrast, results obtained with MEKC at pH 11.0 proved to be anologous to those obtained by RP‐HPLC, with highly hydrophobic peptides – migrating slower than peptides without hydrophobic moieties.
Two‐dimensional electrophoresis of plant proteins and standardization of gel patterns
Authors: Tsugita Akira , Kamo Masaharu , Kawakami Takao , Ohki Yuta 
Published: May 2005

AbstractProteins of two plants, Arabidopsis thaliana and rice (Oryza sativa) were subjected to two‐dimensional electrophoresis analysis with two modifications: (i) comigration of external standard marker proteins with resultant horizontal and vertical stripes in the gel, and (ii) deblocking with a vapor of aqueous heptafluorobutyric acid for N‐acetylserine. Approximately 5000 protein spots were separated from both the five tissues of Arabidopsis and the nine tissues of rice. Over one hundred spots were electroblotted for N‐terminal sequencing. Among the newly sequenced proteins, 62 were from Arabidopsis and 51 from rice.
Characterization of human serum amyloid A protein isoforms separated by two‐dimensional electrophoresis by liquid chromatography/electrospray ionization tandem mass spectrometry
Authors: Ducret Axel , Bruun Cathrine Foyn , Bures Edward J. , Marhaug Gudmund , Husby Gunnar , Aebersold Ruedi 
Published: May 2005

AbstractA detailed structural analysis of the serum amyloid A proteins (SAA) of an individual with highly active, chronic rheumatoid arthritis is reported. SAA isoforms were separated by high‐resolution two dimensional (2‐D) gel electrophoresis. Peptide mapping by reverse‐phase chromatography/electrospray ionization tandem mass spectrometry was applied to correlate the protein(s) contained in each spot with their respective coding gene and to study the posttranslational processing and modification events which might result in differential electrophoretic mobility. Nine protein spots were analyzed. The six major spots corresponded to the Arg and des‐Arg forms of SAA1α and SAA2α, respectively, and to the glycosylated and nonglycosylated form of constitutive serum amyloid A protein (C‐SAA). Two minor spots were identified as SAA1α isoforms containing post‐translational modifications. We suggest that these variants contained a γ‐N,N′‐dimethylasparagine residue at position 83 and that one of them was additionally oxidized at Trp53 and Trp85. The ninth spot was shown to contain a mixture of SAA1α and SAA2α. To our knowledge, this is the first report in which analysis of peptides has been used to verify the presence of C‐SAA in acute‐phase serum. Furthermore, the data illustrate that extensive post‐translational processing results in a structurally diverse class of acute‐phase SAA proteins, which are derived from a small number of genes. Finally, the fast and conclusive technology used in this study promises to be generally useful for the comprehensive investigation of proteins at the level of the primary structure.
Application of combined mass spectrometry and partial amino acid sequence to the identification of gel‐separated proteins
Authors: Patterson Scott D. , Thomas Didier , Bradshaw Ralph A. 
Published: May 2005

AbstractThe combined use of peptide mass information with amino acid sequence information derived by chemical sequencing or mass spectrometry (MS)‐based approaches provides a powerful means of protein identification. We have used a two‐part strategy to identify proteins from nerve growth factor (NGF)‐stimulated rat adrenal pheochromocytoma cell line PC‐12 cell lysates that associate with the adaptor protein Shc (Shc homologous and collagen protein). Initial experiments with metabolically radiolabeled cell extracts separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) revealed a number of proteins that coimmunoprecipitated with anti‐Shc antibody compared with control (unstimulated) cell extracts. The experiment was scaled up and cell lysate from NGF‐stimulated PC‐12 cells was applied to a glutathione‐S‐transferase (GST)‐Shc affinity column, eluted, separated by SDS‐PAGE and blotted to Immobilon‐CD. The blotted proteins were proteolytically digested in situ, and the masses obtained from the extracted peptides were used in a peptide‐mass search program in an attempt to identify the protein. Even if a strong candidate was found using this search, an additional step was performed to confirm the identification. The mixtures were fractionated by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and subjected to chemical sequencing to obtain (partial) sequence information, or post‐source decay (PSD‐) matrix‐assisted laser‐desorption ionization (MALDI)‐MS to obtain sequence‐specific fragment ions. This data was used in a peptide‐sequence tag search to confirm the identity of the proteins. This combined approach allowed identification of four proteins of Mr 43000 to 200000. In one case the identified protein clearly did not correspond to the radiolabeled band, but to a protein contaminant from the column. The advantages and pitfalls of the approach are discussed.
Enhanced in situ gel digestion of electrophoretically separated proteins with automated peptide elution onto mini reversed‐phase columns
Authors: Eckerskorn Christoph , Grimm Rudolf 
Published: May 2005

AbstractAn improved method for the generation and automated isolation of internal peptides by in situ gel digestion of electrophoretically separated proteins is described [1]. To enhance the sensitivity of the method, and to reduce the amount of sample handling steps, we have automated the extraction procedure of peptides after protein cleavage in a sodium dodecyl sulfate (SDS) gel matrix. The excised protein‐containing polyacrylamide bands or spots are first minced to defined particles of about 30 μm. After in situ gel digestion, the gel slurry is transferred into a mini reversed‐phase column‐funnel assembly in the sample loading station of the Hewlett‐Packard protein sequencer. Applying nitrogen pressure elutes peptides from the gel slurry onto the reversed‐phse material. The mini reversed‐phase column is then placed in an in‐line column adapter and connected to a micropreparative high performance liquid chromatography (HPLC) column, where separation of the peptides under standard conditions is achieved. In the work described here complete digestions and excellent peptide recoveries allowed the generation of extensive internal sequence information from low picomole amounts of proteins. The method has been routinely applied in both laboratories for two yearsPart of the results were presented as a poster at the Protein Society Meeting 1994 in San Diego.
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Use of excluded volume to increase the heterogeneity of pore size in agarose gels
Authors: Serwer Philip , Harris Renee A. , Miller Margaret M. , Griess Gary A. 
Published: May 2005

AbstractWhen testing theoretical models that quantitatively describe the sieving of macromolecules during gel electrophoresis, investigators have been limited by absence of control of the heterogeneity of the size of pores in the gel. In a recent study performed by electron microscopy of thin sections (G. A. Griess et al., J. Struct. Biol. 1993, 111, 39–47), pore size heterogeneity has been increased for agarose gels by a combination of both derivatization and molecular weight reduction of the polysaccharide chains of agarose. In the present study, pore size heterogeneity is increased by a mechanism that appears to have an origin different from the origin of this previously observed increase in heterogeneity: Pore size heterogeneity is increased by addition of a polyethylene glycol (PEG) of high molecular weight (18 500) to molten agarose before gelation. In contrast, the use of a lower molecular weight PEG (either 4 000 or 7 500) causes the formation of micron‐sized precipitates within a gelled network of agarose fibers. Thus far, the PEG‐induced heterogeneity of pore size occurs primarily in 100–1000 μm scale zones separated from each other by interzone regions of decreased agarose fiber density. More uniform gels are needed for the study of sieving.
An exactly solvable Ogston model of gel electrophoresis: I. The role of the symmetry and randomness of the gel structure
Authors: Slater Gary W. , Guo Hong L. 
Published: May 2005

AbstractThe Ogston‐Morris‐Rodbard‐Chrambach model (OMRCM) of gel electrophoresis assumes that the mobility (μ) of charged particles is directly proportional to the fractional volume (f) of the gel that is available to them. Many authors have studied the fractional volume f in detail for various particle shapes, but the original assumption, that μsf, has not been scrutinized seriously. In fact, this geometrical model of electrophoresis does not take into account the connectivity of the gel pores or the precise gel architecture. Recently (G. W. Slater and H. L. Guo, Electrophoresis 1995, 16, 11–15) we developed a Monte Carlo computer simulation algorithm to study the electrophoretic motion of simple particles in gels in the presence of fields of arbitrary strength. Our preliminary results indicated that the mobility and the fractional volume were not generally proportional to one another. In this article, we show how to calculate, in the limit where the field intensity is vanishingly small, the exact electrophoretic mobility of particles in any type of gel in two or more dimensions. Our results, presented here for some simple two‐dimensional systems, indicate that a particle can have different electrophoretic mobilities in gels in which it has access to the same fractional available volume f. The curvature of the Ferguson plot is shown to be related to the symmetry and the degree of randomness that characterize the gel. We also demonstrate that the OMRCM is, in fact, a mean field approximation that corresponds to a uniform, annealed gel. We thus conclude that the relation between the electrophoretic mobility and the gel concentration (C) is a delicate function of the gel architecture, and that one needs more than the fractional volume f to fully characterize the transport properties of migrating particles in separation media. Exact relationships between the mobility μ and the gel concentration C are given for our model gels.
A gel as an array of channels
Authors: Zimm Bruno H. 
Published: May 2005

AbstractWe consider the theory of charged point molecules (‘probes’) being pulled by an electric field through a two‐dimensional net of channels that represents a piece of gel. Associated with the position in the net is a free energy of interaction between the probe and the net; this free energy fluctuates randomly with the position of the probe in the net. The free energy is intended to represent weak intractions between the probe and the gel, such as entropy associated with the restriction of the freedom of motion of the probe by the gel, or electrostatic interactions between the probe and charges fixed to the gel. The free energy can be thought of as a surface with the appearance of a rough, hilly landscape spread over the net; the roughness is measured by the standard deviation of the free‐energy distribution. Two variations of the model are examined: (1) the net is assumed to have all channels open, or (2) only channels parallel to the electric field are open and all the cross‐connecting channels are closed. Model (1) is more realistic but presents a two‐dimensional mathematical problem which can only be solved by slow iteration methods, while model (2) is less realistic but presents a one‐dimensional problem that can be reduced to simple quadratures and is easy to solve by numerical integration. In both models the mobility of the probe decreases as the roughness parameter is increased, but the effect is larger in the less realistic model (2) if the same free‐energy surface is used in both. The mobility in model (2) is reduced both by high points in the rough surface (‘bumps’) and by low points (‘traps’), while in model (1) only the traps are effective, since the probes can flow around the bumps through the cross channels. The mobility in model (2) can be made to agree with model (1) simply by cutting off the bumps of the surface. Thus the simple model (2) can be used in place of the more realistic model (1) that is more difficult to compute.
The limiting mobility of DNA sequencing fragments for both cross‐linked and noncross‐linked polymers in capillary electrophoresis: DNA sequencing at 1200 V cm−1
Authors: Yan Juying , Best Norine , Zhang Jian Zhong , Ren Hongji , Jiang Rong , Hou Joan , Dovichi Norman J. 
Published: May 2005

AbstractThe mobility of DNA sequencing fragments was measured in Long‐Ranger gels at an electric field ranging from 200 to 1200 V cm−1 and in noncross‐linked polyacrylamide at electric fields ranging from 100 to 300 V cm−1. In both cases, N*, the fragment length that denotes the onset of biased reptation with orientation, is inversely proportional to electric field. The inverse dependence of N* is inconsistent with the original biased reptation model but is consistent with modern models of DNA migration. While separation speed increases dramatically with electric field, the number of bases determined in a separation decreases in proportion to field strength. We present a DNA sequencing run at an electric field of 1200 V cm−1. Roughly 200 bases of sequence are determined in 3.5 min.
Temperature profile of buffer‐filled electrophoresis capillaries using air convection cooling
Authors: Nishikawa Tetsuo , Kambara Hideki 
Published: May 2005

AbstractTemperature profiles of buffer‐filled capillaries (capillary temperature), using both natural and forced air convection for cooling, were obtained both theoretically and experimentally in order to achieve stable capillary electrophoresis for accurate DNA sequencing analysis. The capillary temperature was calculated using the temperature dependency of the buffer resistivity, and the threshold values of the electric field and the capillary temperature for temperature instability were characterized. The capillary temperature became unstable at about 420 V/cm and at about 62°C regardless of the outer diameter of the capillary; these values approximately coincide with the measured values obtained using a very thin thermocouple. The sets of values of electric field, air temperature, and air flow velocity in forced air cooling were obtained to keep the capillary temperature at the 40 ∼ 60°C needed for accurate DNA sequencing in a Tris‐boric acid‐EDTA buffer.
Resolution of apolipoprotein B repeat unit position variants on agarose, denaturing, and native polyacrylamide gels
Authors: Prinz Mechthild , Schmitt Cornelia , Staak Michael , Baum Howard 
Published: May 2005

AbstractUtilizing three different electrophoresis systems for the separation of variable number of tandem repeats (VNTR) alleles of the high variable region 3′ from the apolipoprotein B (Apo B) locus, it could be shown that apparent allele subtypes on native polyacrylamide gels comigrated with the regular ladder alleles on agarose and denaturing polyacrylamide gels. Two subtypes, one with a shift towards the cathode and one shifted towards the anode, and the corresponding ladder alleles were sequenced in order to determine the molecular basis for this aberrant electrophoretical mobility. Subtype and ladder alleles displayed the same number of repeat units but differed in the number and order of different types of repeats. Depending on the variation in the region of pure AT repeat units, the potential secondary structures of hairpin loop formation differed for subtypes and ladder alleles, which is thought to explain the shift in electrophoretic mobility on native polyacrylamide gel electrophoresis.
Isozymes of a rattan species and their genetic interpretation
Authors: Bon Marie‐Claude 
Published: May 2005

AbstractA combination of a modified Feret' (Silvae Genet. 1971, 20, 46–50) extraction buffer and two types of electrophoresis with acrylamide and starch gels were used to characterize allozymes in mature vegetative tissue of a commercially high value species of rattans (Calamus subinermis). From the analysis of allelic segregation from single maternal rattans and their offspring, genetic control of the 16 observed banding zones, which were consistently scorable, was assumed. Seventeen gene loci were identified. The percentage of polymorphic loci within Calamus subinermis was much higher (70.5%) than expected levels of genetic diversity for tropical woody and non‐woody species. It is thought that the protocol described may be applied to the analysis of the genetic diversity of all the endangered Calamus species.
Interpretation of electrophoretic band shapes by a partition chromatographic model
Authors: Weiss George H. , Sokoloff Helen , Zakharov Sergey F. , Chrambach Andreas 
Published: May 2005

AbstractMeasurements of the shape of electrophoretic bands of phycoerythrin and conalbumin have been made at regular intervals during migration in agarose gels. Analysis of the peak shapes suggests the existence of a significant degree of asymmetry. This is to be contrasted with the symmetry around the peak associated with the generally assumed Gaussian band. The degree of asymmetry of the bands decreased as a function of time and increased with agarose concentration. A similar experiment on DNA indicated constancy of the degree of asymmetry as a function of time. These results can be interpreted as, but do not prove the validity of, a nonlocal diffusion equation which generalizes a theory originally put forth by Giddings and Eyring (J. Am. Chem. Soc. 1955, 59, 416–420). The results may be significant in framing a measure of the resolvability of electrophoretic peaks.
Analysis of macromolecular branched chain polypeptides by capillary electrophoresis and micellar electrokinetic chromatography
Authors: Idei Miklós , Dibó Gábor , Bogdán Katalin , Mezö Gábor , Horváth Anikó , Érchegyi Judit , Mészáros György , Teplán István , Kéri György , Hudecz Ferenc 
Published: May 2005

AbstractAmphoteric poly(Lys‐[Glu1.0‐DL‐Ala4.1]), (EAK) and anionic poly(Lys‐Ac‐Glu0.98‐DL‐Ala3.98]), (AcEAK) branched chain polypeptides were analyzed by capillary electrophoresis (CE) and micellar elektrokinetic chromatography (MEKC) in the following buffers. A1: 0.25 N triethyl ammonium phosphate (TEAP) buffer (pH 2.25); A2: 100 mM sodium dodecyl sulfate (SDS) in buffer A1; B1: Na‐borate buffer (pH 7.7); B2: 100 mM SDS in buffer B1; C1: Na‐borate buffer (pH 11.0); C2: 100 mM SDS in buffer C1. Both EAK and AcEAK could be separated by a CE mechanism at pH 2.25 and by an MEKC mechanism at pH 11.0. Optimum results were achieved with CE in buffer A1 and with MEKC in buffer C2.