bluuebunny/crossref_metadata_2025 huggingface dataset:
A World Wide Web (WWW) server database engine for an organelle database, MitoDat
Authors: Lemkin Peter F. , Chipperfield Michael , Merril Carl , Zullo Steven
Published: May 2005

We describe a simple database search engine “dbEngine” which may be used to quickly create a searchable database on a World Wide Web (WWW) server. Data may be prepared from spreadsheet programs (such as Excel, etc.) or from tables exported from relational database systems. This Common Gateway Interface (CGI‐BIN) program is used with a WWW server such as available commercially, or from National Center for Supercomputer Algorithms (NCSA) or CERN. Its capabilities include: (i) searching records by combinations of terms connected with ANDs or ORs; (ii) returning search results as hypertext links to other WWW database servers; (iii) mapping lists of literature reference identifiers to the full references; (iv) creating bidirectional hypertext links between pictures and the database. DbEngine has been used to support the MitoDat database (Mendelian and non‐Mendelian inheritance associated with the Mitochondrion) on the WWW.
Identification of proteins by their amino acid composition: An evaluation of the method
Authors: Golaz Olivier , Wilkins Marc R. , Sanchez Jean‐Charles , Appel Ron D. , Hochstrasser Denis F. , Williams Keith L.
Published: May 2005

Expression of different genomes can be studied by high‐resolution two‐dimensional electrophoresis (2‐D PAGE). To help these studies, two‐dimensional reference maps of different biological tissues and fluids have been built and can be found in the SWISS‐2DPAGE database, accessible via the World Wide Web network on the ExPASy molecular biology server. Different techniques were used to identify the polypeptides. At the present time, the method considered to be the fastest and the most cost‐effective is amino acid composition analysis (AAC). Proteins, transferred onto polyvinylidene (PVDF) membranes, were submitted to vapor‐phase hydrolysis, derivatized with 9‐fluorenylmethyl chloroformate (FMOC) and separated on an ODS‐Hypersil column. Identification was obtained by using the program ‘AACompIdent’ available from ExPASy. In this work, different experimental parameters, such as contamination, reproducibility and accuracy, have been assessed. First, it has been found that a major source of contamination was human keratin. Next, amino acids have been classified into ‘reliable’ and ‘nonreliable’. Accordingly, ‘bias’ and ‘weights’ were defined for each amino acid, which could be set in the ‘AACompIdent’ program. Finally, examples of identification, including the use of Edman degradation sequence tagging, are described.
Characterisation of proteins from two‐dimensional electrophoresis gels by matrix‐assisted laser desorption mass spectrometry and amino acid compositional analysis
Authors: Wheeler Colin H. , Berry Sophie L. , Wilkins Marc R. , Corbett Joseph M. , Ou Keli , Gooley Andrew A. , Humphery‐Smith Ian , Williams Keith L. , Dunn Michael J.
Published: May 2005

Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two‐dimensional electrophoresis (2‐DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2‐DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2‐DE gels.
Identification of different galectins by immunoblotting after two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients
Authors: Lutomski Didier , Caron Michel , Cornillot Jean‐Denis , Bourin Philippe , Dupuy Catherine , Pontet Michel , Bladier Dominique , Joubert‐Caron Raymonde
Published: May 2005

Vertebrate soluble β‐galactoside‐binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross‐reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), even when followed by immunoblottin. However, their different isoelectric points allow their identification using isoelectric focusing and two‐dimensional (2‐D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two‐dimensional electrophoresis with immobilized pH gradient (IPG‐Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2‐D immunoblotting using anti‐galectin 1 (Gal‐1) and anti carbohydrate‐binding protein 15 (CBP15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG‐Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside‐binding proteins even at basic p/s.
Plasma protein adsorption patterns on liposomes: Establishment of analytical procedure
Authors: Diederichs Julia E.
Published: May 2005

After intervenous (i.v.) injection, colloidal drug carriers such as liposomes, emulsions, polymeric or solid lipid nanoparticles immediately interact with plasma proteins. The adsorbed plasma protein patterns depend on physicochemical characteristics of the carriers' surface and are regarded as a key factro for the in vivo behavior of the carriers. The comprehension of the correlation between protein adsorption and in vivo organ distribution can be utilized to obtain drug targeting to different tissues. Carriers with different protein adsorption patterns will interact with different tissue‐specific receptors or will be recognized by different macrophage subpopulations. Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was applied to determine the protein adsorption pattern on polystyrene particles as model carriers. To transfer this analytical method to i.v. injectable colloidal carriers such as liposomes, a new sample preparation method was developed. The separation of liposomes from plasma after incubation was achived by gel filtration using a Sepharose 2B column. This technique allowed a mild separation independent from eluent composition and only according to size differences. Possible protein desorption from the liposomes and adsorption onto the gel were minimized by using an eluent with a sufficiently high ionic stregth. To estimate the efficiency of separation, the content of liposomes and plasma in each fraction being eluted was determined by ultraviolet (UV) spectroscopy. With this new separation method plasma protein adsorption patterns on liposomes could be analyzed for the first time. The sample preparation by gel filtration seemed to have no influence on liposome stability as far as size distribution is concerned.
Pulsed‐field trapping electrophoresis: A computer simulation study
Authors: Desruisseaux Claude , Slater Gary W.
Published: May 2005

Experimental investigations have shown that adding a large, globular and neutral protein (such as streptavidin) at one end of the DNA fragments to be separated by gel electrophoresis strongly affects the dynamics of these molecules, leading to what is known as trapping electrophoresis (TE). In TE, the velocity decreases much more rapidly with DNA molecular size than under normal gel electrophoresis conditions, suggesting that TE may be used to increase the power of separation of polyacrylamide gel electrophoresis. Unfortunately, the bands are broader and fewer readable bands can fit on a single gel slab. Our previous theoretical study of TE also predicted the existence of long‐lasting anomalous regimes where one cannot define a velocity or a diffusion constant. These secondary effects of trapping are related to the very broad distribution of detrapping times (the time needed to exit a trap). In order to increase the usefulness of TE, it has been suggested that pulsed fields may help the molecules exit traps more rapidly. In this article, we present a detailed numerical study of pulsed field TE. We conclude that simple pulsed fields alone may not be enough to increase the sequencing power of polyacrylamide TE because the rate of band broadening cannot be controlled. We also report the existence of anomalous regimes in the presence of pulsed fields, a factor that has been previously neglected in analytical models. Other approaches are also proposed.
Influence of optical probing with YOYO on the electrophoretic behavior of the DNA molecule
Authors: Carlsson Christina , Larsson Anette , Jonsson Mats
Published: May 2005

The influence of the fluorescent dye YOYO (1,1′‐(4,4,8,8,‐tetramethyl‐4,8‐diazaundecamethylene)bis[4‐[[3‐methyl‐benzo‐1,1‐oxazol‐2‐yl]methylidene]‐1,4‐dihydroquinolinium] tetraiodide) on the electrophoretic behavior of the DNA molecule was investigated. This is important when using YOYO as a probe in capillary electrophoresis or in fluorescence microscopy studies of DNA with the purpose of studying the migration mechanism of DNA on the molecular level. We have measured the mobility and orientation dynamics (using the linear dichroism technique) for both pure DNA and the YOYO‐DNA complex in agarose gel in order to compare their electrophoretic properties. Mobility decreases, the degree of orientation becomes lower, and the orientational dynamics slower, when YOYO binds to DNA. However, the dependence on field strength of the mobility, orientation and orientational dynamics, are similar for DNA and YOYO‐DNA, indicating that the mode of migration does not change significantly upon binding YOYO to DNA. Furthermore, since our results show that the effect of YOYO on both the degree of orientation and orientational dynamics of the DNA can be measured and therefore be compensated for, it can be concluded that YOYO is an excellent optical probe for the study of the migrational behavior of DNA.
An analysis of the dynamic range and linearity of an infrared DNA sequencer
Authors: McIndoe Richard A. , Hood Leroy , Bumgarner Roger E.
Published: May 2005

We are currently developing genotyping software and protocols for use on the Li‐Cor model 4000(L) infrared fluorescence DNA sequencers. During the development of the genotyping software it became apparent that the potential dynamic range of the instrument was not being realized when the data collection parameters were optimized to produce a high‐contrast image on the computer screen. In particular, we were unable to obtain peaks with signal to noise (S/N) greater than about 30:1 and the stronger peaks often saturated the detector. Because the numerical dynamic range available in 16‐bit data collection mode exceeds 65000, our limited dynamic range of about 30 in actual data was somewhat puzzling. Hence, we undertook a study to explore the dynamic range and linearity of the Li‐Cor DNA sequencer in order to minimize background fluorescence and noise as well as maximize the available S/N. Data is presented on the background and noise using different polyacrylamide gel mixes with various signal gain and offset values, and the relative contribution of the glass, gel and instrumentation to the background fluorescence is discussed. Based on these results, optimum gain and offset values were determined that maintain the background fluorescence at approximately 1–2% of the maximum dynamic range with a minimum amount of noise. Using these optimized values, we determined the detection system is linear over three orders of magnitude by titrating known quantities of an infrared fluorophore‐labeled primer. In addition, we were able to detect approximately 15.2 amoles of labeled primer. The results provided by this study establish a set of guidelines for evaluating how to set the signal gain and offset in order to minimize the background and maximize the S/N and dynamic range of the Li‐Cor sequencer.
Precast commercial polyacrylamide gels for separation of DNA amplificates by temperature gradient gel electrophoresis: Application to clonality analysis of lymphomas
Authors: Suttorp Meinolf , Von Neuhoff Nils , Tiemann Markus , Dreger Peter , Schaub Jürgen , Löffer Helmut , Parwaresch Reza , Schmitz Norbert
Published: May 2005

The third complementary determining region (CDR‐III) of the rearranged immunoglobulin heavy chain (IgH) genes represents a unique marker for a lymphocyte and its clonal descendants and can be amplified by the polymerase chain reaction (PCR) technique. This approach has markedly enhanced the sensitivity for detection of clonal lymphocyte populations in patients with malignant B‐lymphoid neoplasias. To monitor minimal residual disease (MRD) in tissue specimens during or after antineoplastic treatment, the problem of detecting the presence of a few clonal (malignant) lymphocytes in coexistence with a majority of polyclonal lymphocytes has to be addressed. Semi‐nested PCR amplification of CDR‐III rearrangements from specimen infiltrated by tumor cells generates clonal signals in front of a polyclonal background, and therefore high resolution electrophoretic techniques for separation of DNA fragments are required. Temperature gradient gel electrophoresis (TGGE) resolving DNA homo‐ and heteroduplexes according to their thermal stability has been successfully applied for this purpose using special electrophoretic equipment. We describe an adjustment to this technique by using a commercially available precast 0.5 mm thick polycarylamide gel and by changing a standard horizontal electrophoretic device into a TGGE device. By this means we screened patients with B‐cell lymphoma undergoing high‐dosage radiochemotherapy followed by autologous transplantation for continuous presence of clonal (tumor‐specific) CDR‐III rearrangements. Specimens from blood and bone marrow were collected on diagnosis as well as before and after autologous transplantation. In addition, the autograft (bone marrow or peripheral blood hematopoietic stem cells) was analyzed. Tumor cells were easily detected in the transplants and in specimens collected during follow‐up examinations. The clinical value of these findings remains unclear as yet because the number of cases investigated was small and the follow‐up time is still too short. However, we conclude that the technique of combining the sensitivity of PCR with the specificity of high resolution TGGE is easy to use, making it possible to handle, in a clinical routine, a great number of samples within a short time in order to monitor MRD in patients with B‐cell neoplasias.
High resolution slab gel electrophoresis of 8‐amino‐1,3,6‐pyrenetrisulfonic acid (APTS) tagged oligosaccharides using a DNA sequencer
Authors: O'Shea Michael G. , Morell Matthew K.
Published: May 2005

A novel electrophoretic method for the analysis of oligosaccharides using DNA sequencer technology is illustrated using malto‐oligosaccharide distributions obtained following isoamylase disgestion of glycogen, wheat starch and potato starch. The debranched starches were derivatized at the reducing end with the charged fluorophore 8‐amino‐1,3,6‐pyrenetrisulfonic acid (APTS). This highly reproducible method provides baseline resolution of oligomers from chain lengths of 3 to more than 80 glucose units, and exhibits high sensitivity with detection thresholds of one femtomole per resolved band. In addition, the reductive amination procedure attaches a single fluorophore per oligosaccharide, allowing calculation of the results on either a mass or a molar basis. The efficacy of the method is illustrated through the determination of the profile of individual oligosaccharides of chain length with a degree of polymerization (DP) < 80, derived from loading less than 15 ng per analysis of glycogen, wheat and potato starches. While the results obtained were superior in resolution and sensitivity to previously reported observations using a range of techniques, they were nonetheless consistent with the overall differences between these polysaccharides. The resolution, sensitivity, reproducibility and high throughput of the method provides substantial advantages over existing methods for the analysis of linear oligosaccharide chain length distributions.
Pyrogallol red‐molybdate: A reversible, metal chelate stain for detection of proteins immobilized on membrane supports
Authors: Shojaee Negin , Patton Wayne F. , Lim Mark J. , Shepro David
Published: May 2005

Certain metal complexes selectively interact with proteins immobilized on solid‐phase membrane supports to form brightly colored products. The metal chelates form protein‐dye complexes in the presence of metal ions at acidic pH but are eluted from the proteins by immersing membranes in a solution of basic pH that contains other chelating agents. The reversible nature of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, N‐terminal and internal protein sequencing. Among the metal complexes evaluated to date, the triazine dye‐ferrous complexes (ferene S, ferrozine) and the ferrocyanide‐ferric complexes provide the most sensitive detection of proteins immobilized on membranes. While the pyrogallol redmolybdate complex is commonly used in solution‐based total protein assays, its utility as a reversible stain for proteins immobilized on membranes has not been reported. Pyrogallol red‐molybdate complexes readily stain proteins on nitrocellulose and polyvinyl difluoride membranes with similar sensitivity as ferrozine‐ferrous complexes. Analysis of charge‐fractionated carrier ampholytes and synthetic polymers of different L‐amino acids indicate that binding is prominently via protonated α and ε‐amino side chains. Carbamylation of amino groups in bovine serum albumin substantially diminishes pyrogallol red‐molybdate binding to the protein. The stain is reversible, resistant to chemical interference, and compatible with immunoblotting.
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

A 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

The 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

Capillary 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

Proteins 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

A 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

The 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

An 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 years

Part of the results were presented as a poster at the Protein Society Meeting 1994 in San Diego.

.
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

When 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

The 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

We 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

The 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

Temperature 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

Utilizing 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

A 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

Measurements 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

Amphoteric 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.
Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide gel electrophoresis
Authors: Michaud Dominique , Cantin Line , Bonadé‐Bottino Michel , Jouanin Lise , Vrain Thierry C.
Published: May 2005

The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla. M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second‐stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans‐epoxysuccinyl‐L‐leucylamido‐(4‐guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class‐specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition by OCI was partly prevented during electrophoresis. This species‐related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne.
Amino acid analysis of proteins separated by two‐dimensional electrophoresis in maize: Isoform detection and function identification
Authors: Touzet Pascal , de Vienne Dominique , Huet Jean‐Claude , Ouali Christian , Bouet Françoise , Zivy Michel
Published: May 2005

The possibility of using experimentally determined amino acid composition to assess relatedness between 75 proteins separated by two‐dimensional electrophoresis (2‐DE) and to identify them was tested on maize. Two independent parameters, the relative Euclidean distance and the correlation coefficient between the amino acid compositions, were evaluated and used. Previous sequence information for 31 out of the 75 proteins made it possible to evaluate the method for the detection of isoforms and for identification. However, the extension of the interrogation beyond maize to all plant sequences raised the problem of false positives that could nevertheless be limited by replications and by using additional information. The efficiency of the method to assess relatedness betwen proteins should make amino acid composition analysis a valuable tool in large protein characterization programs based on 2‐DE, by facilitating the transfer of information from one well‐documented organ/tissue or genotype to another.
High‐resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy‐transfer fluorescent primers
Authors: Wang Yiwen , Wallin Jeanette M. , Ju Jingyue , Sensabaugh George F. , Mathies Richard A.
Published: May 2005

Short tandem repeat regions (STRs) from the polymorphic loci VWFA, THO1, TPO and CSF were amplified by the multiplex polymerase chain reaction (PCR) and analyzed by capillary array electrophoresis with fluorescence detection of energy transfer (ET) labels. The fluorescent ET primers are labeled with one fluorescein at the 5′ end and a second fluorescein at the position of the 7th or 9th (modified) base to produce fragments that fluoresce in the green (λmax = 525 nm). M13 A‐track sequencing fragments, used as an internal sizing standard, were generated with a universal primer that has a donor fluorescein at the 5′ end and a rhodamine acceptor at the position of the 11th (modified) base to produce fragments fluorescing in the red (> 590 nm). The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two‐color confocal fluorescence scanner. Separations were performed on arrays of hollow fused silica capillaries filled with denaturing and replaceable hydroxyethyl cellulose sieving matrices. Separations were complete in less than 50 min, and single base resolution as well as reproducible STR sizing was achieved. The relative standard deviation in sizing was below 0.6%. This work establishes the feasibility of high‐resolution, high‐speed and high‐throughput STR typing of single‐stranded DNA fragments using capillary array electrophoresis.
Characterization of mitochondrial DNA using lowstringency single specific primer amplification analyzed by laser induced fluorescence – capillary electrophoresis
Authors: Marino Michael A. , Weaver Kristal R. , Tully Lois A. , Girard James E. , Belgrader Phillip
Published: May 2005

Polymerase chain reaction (PCR)‐based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single nucleotide polymorphisms (SNPs) require other biochemical reactions in addition to PCR to identify the sequence polymorphisms. Low‐stringency sequence‐specific PCR (LSSP‐PCR) is an example of a recent method that does not require additional biochemical treatments. The analysis of LSSP‐PCR by capillary electrophoresis (CE) to discriminate the highly polymorphic mitochondrial DNA (mtDNA) D‐loop region is described. The DNA from five individuals were amplified (first step) using sequence‐specific primers to produce 1021 bp fragments containing the D‐loop region. Each fragment was isolated by electroelution using CE and UV detection, and subjected to a second amplification (second step) using a single primer annealed under low stringency conditions. This generated a range or profile of PCR products for each sample, which were resolved and analyzed by CE with the intercalator TOTO‐1 and laser‐induced fluorescence (LIF) detection. The LSSP‐PCR profiles were unique for each individual, indicating that this technique may be applicable for forensic identity testing.