bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of nonparallel alternating fields on the mobility of DNA in the biased reptation model of gel electrophoresis
Authors: Slater Gary W. , Noolandi Jaan 
Published: May 2005

AbstractChromosome‐size DNA molecules can now be separated using a variety of pulsd field gel electrophoresis techniques. In this article, we study the predictions of the biased reptation model concerning the effect of two pulsed fields, making an arbicray angle, on the power of separation of gel electrophoresis. Separation is predicted to be largely enhanced for obtuse angles, in agreement with experiments. Interestingly very large molecules, which are not separated by pulsed fields, are predicted not to migrate along the gel diagonal for fairly long periods of time. Finally, we discuss the optimization of these techniques using the results of the theory, and the limitations of the latter when fluctuations and intramolecular modes probably dominate the system.
Monte Carlo simulation of DNA electrophoresis
Authors: Batoulis Jannis , Pistoor Norbert , Kremer Kurt , Frisch Harry L. 
Published: May 2005

AbstractThis paper describes an attempt to study the electrophoresis mobility of a DNA molecule in a gel by means of a Monte Carlo simulation. We find that the electrophoresis mobility μ can be well described by the empirical equation μ = κ1/N + κ2E2 with N being the number of monomers of the model chain and E being the applied field. For small E the data can merge into the linear response result μ = κ1/N. The paper also discusses necessary extensions of the present approach.
The application of high resolution two‐dimensional polyacrylamide gel electrophoresis to the identification and purification of a protein, NG 8.4, present in Neisseria gonorrhoeae and the subsequent development of a radioimmunoassay
Authors: Jackson Peter , Urwin Valerie E. , Torrance Michaela R. , Carmen J. Anthony 
Published: May 2005

AbstractA soluble protein of unknown function was shown, by two‐dimensional polyacrylamide gel electrophoresis, to be present in Neisseria gonorrhoeae at significantly higher concentrations than in the other related bacteria tested. The data indicate the possibility of this protein being specific to Neisseria gonorrhoeae. This protein was designated as NG 8.4 and purified. A radioimmunoassay was developed for the measurement of this protein and subsequently used to determine the degree of cross reaction exhibited by a number of species of bacteria. Of the bacteria tested only those of the genus Neisseria gave a significant reaction in the assay.
Isoelectric focusing on cellulose acetate membranes: An up‐date on materials and methods
Authors: Ambler Jeffrey 
Published: May 2005

AbstractThe method of isoelectric focusing proteins on cellulose acetate membranes has been improved by the introduction of: (i) plastic‐backed membranes, (ii) a commercially available Mini‐IEF cell and (iii) sensitive colloidal gold staining. The improved methodology is described with applications for clinical laboratories.
Repel‐Silane induced alteration of group specific component subtypes using LKB immobiline DryPlates
Authors: Klein Hartmut , Stiefel Katrin 
Published: May 2005

AbstractGroup specific component (GC) band patterns of sensitive blood samples may be altered by Repel‐Silane. Alterations occur when isoelectric focusing is performed with LKB Immobiline DryPlates rehydrated in a reswelling cassette using Repel‐Silane as a hydrophobic additive. In two cases Repel‐Silane converted common GC subtypes into apparent rare types.
Heterogeneity of serum creatine kinase isoenzyme‐MM in acute myocardial infarction
Authors: Williams John , Williams Katherine M. , Märshall Thomas 
Published: May 2005

AbstractIsoelectric focusing of serum creatine kinase (CK; EC 2.7.3.2) reveals up to 14 CK‐MM subbands following acute myocardial infarction (AMI). The “normal” subbands 1 (pI 6.91), 2 (pI 6.65) and 3 (pI 6.35) are faintly present in normal serum and the “abnormal” subbands c (pI 7.25), e (pI 6.85), g (pI 6.50), i (pI 6.28), j (pI 6.20) and k (pI 6.15) are prominently detected in sera with elevated CK. “Abnormal” subbands a (pI 7.55), b (pI 7.35), d (pI 7.05), f (pI 6.72) and h (pI 6.40) have only been detected in AMI. The “abnormal” subbands appear, and reach maximum intensity (together with CK‐MM 1–3), 3–12 h after infarction, and become faint and anodally convert (as do CK‐MM 1–3) within 36 h. Similar changes are detected by nonequilibrium pH gradient electrophoresis which combines CK‐MM and CK‐MB analysis. In vitro incubation of serum with 0.015 M 2‐mercaptoethanol induces conversion of CK‐MM 1, 2 and 3 to b and c, d and e, and f and g, respectively. Thus, the complexity of the patterns is explained by a secondary conversion of “normal” to “abnormal” subbands superimposed upon anodal conversion of CK‐MM 1 → 3. The clinical significance of these findings is discussed.
Cystic fibrosis proteins detected by electrophoretic techniques
Authors: Guy‐Crotte Odette , Barthe Catherine , Figarella Catherine 
Published: May 2005

AbstractFor the detection of the cystic fibrosis protein (CFP) in serum of cystic fibrosis (CF) carriers, thin‐layer polyacrylamide gel isoelectric focusing proved inappropriate as diagnostic test, but was useful for screening fractions on purification of CFP by chromatofocusing on a Mono P column. On sodium dodecyl sulfate‐polyacrylamide gel electrophoresis an Mr 12 000 protein (P 12) was found in most CFP‐positive sera, indicating good correlation between these two CF‐associated proteins. Detection of the P 12 protein by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was well reproducible and less delicate than IEF. The technique was also used to purify P 12 from serum by two successive preparative electrophoresis steps in a 7.5–15 % gradient and 15 % homogeneous gel. The use of silver staining revealed that P 12 which was present in all sera of CF patients and carriers with variable intensities, was also present in trace amounts in normal sera.
Multiple immunoblot: A sensitive technique to stain proteins and detect multiple antigens on a single two‐dimensional replica
Authors: Steffen Walter , Linck Richard W. 
Published: May 2005

AbstractA multiple immunoblotting technique was developed to positively identify up to three different antigens on a single nitrocellulose replica of a two‐dimensional isoelectric focusing/sodium dodecyl sulfate‐polyacrylamide gel. Three highly sensitive immunoblot assays were selected, including: horseradish peroxidase/luminescence, alkaline phosphatase, and silver‐enhanced immunogold. As a major advantage, the method permits a simultaneous detection of up to three different antigens without eluting the antibody‐dye complex between staining of single polypeptides, thus providing a highly accurate identification of closely migrating components. The staining procedure is summarized in a flow chart. In addition to the multiple immunoblot staining, some suggestions are provided for a sensitive protein staining.
A thin‐layer multistrip polyacrylamide gel electrophoresis apparatus for ferguson plot analysis at the sub‐microgram load level
Authors: Orbán László , Sullivan James V. , Zwieb Christian , Chrambach Andreas 
Published: May 2005

AbstractA procedure was developed for casting thin‐layer multistrip polyacrylamide gels and using them for the simultaneous gel electrophoresis at several gel concentrations (Ferguson plot analysis) at the sub‐microgram load level, using silver staining, autoradiography and, potentially, blotting for detection. The lower viscosity of polymerization mixtures, compared to agarose gelation mixtures, required the redesign of the multistrip cassette with separation of channels by rubber gaskets and the application of a cassette press. The lowered viscosity also required addition of 35% sucrose and an increased rate of polymerization in application to multistrip gels formed on a common NetFix backing. The present design allows one to obtain Ferguson plots exemplified by those of 32P‐labeled DNA followed by autoradiographic detection.
A native, affinity‐based protein blot for the analysis of streptavidin heterogeneity: Consequences for the specificity of streptavidin mediated binding assays
Authors: Dittmer Jürgen , Dittmer Angela , Bruna Roberto Delia , Kasche Volker 
Published: May 2005

AbstractCommercial preparations of streptavidin, a bacterial biotin‐binding protein, were analyzed by isoelectric focusing combined with an affinity‐based protein blot using biotinylated, protein‐saturated nitrocellulose. The colorimetrical detection of strep‐tavidin with biotinylated alkaline phosphatase allows the selective visualization of streptavidin molecules with at least two active biotin‐binding sites. Dependent on the preparation, seven to sixteen streptavidin forms were found with isoelectric points ranging from 5 to 8. Molecular weight analysis of the subunits of streptavidin showed that the observed heterogeneity was mainly due to limited proteolysis, which does not destroy the biotin‐binding activity. The preparations differed also in the nonspecific reactivity of streptavidin with single‐stranded DNA, bovine serum albumin and Tween 20. No relationship was observed between heterogeneity and non‐specific binding activity. Data obtained from protein blots onto nitrocellulose saturated with single‐stranded DNA showed that it cannot be excluded that streptavidin with only a single active biotin‐binding site is mainly responsible for the nonspecific reactivity of some streptavidin preparations.
Step change of counterion – a new option in capillary zone electrophoresis
Authors: Sudor Jan , Stránský Zdeněk , Pospíchal Jan , Deml Mirko , Boček Petr 
Published: May 2005

AbstractIn contrast to common capillary zone electrophoresis, with a constant composition of the background electrolyte, a new method is described, based on a change of the ionic matrix composition during the migration of the sample components. The proposed method expands the scale of pK's of analyzed compounds in a single run and affords new potential for optimizing the separation. The step change of the ionic matrix composition was achieved by displacing the primary matrix by the modified ionic matrix (electrolyte) which migrated against the movement of the sample components. The separation thus proceeded in the primary ionic matrix (electrolyte) while the detection was performed in the modified matrix with resultant shortening of the analysis time and convenient quantitation. A model mixture of five compounds, with a difference of four units in pK could be well resolved.
Formulations for immobilized pH gradients including pH extremes
Authors: Gianazza Elisabetta , Celentano Fabrizio , Magenes Stefania , Ettori Claudia , Righetti Pier Giorgio 
Published: May 2005

AbstractFormulations are given both for narrow (<2 pH units) and for wide range (up to 8 pH units) immobilized pH gradients, spanning between pH 2.5 and pH 11. The contribution from water to the buffering power (β) at these pH extremes requires the recipes to be optimized (in terms of gradient linearity) for each desired level of βav.
Haptoglobin subtype determination by isoelectric focusing in agarose gel: Application to paternity testing and presentation of a new α2‐variant
Authors: Thymann Mariann , Svensmark Ole , Masumba Grethe , Broksø Heidi , Skibsby Lis Bundgaard 
Published: May 2005

AbstractHaptoglobin subtyping of a Danish population sample (n = 2184) by a modified isoelectric focusing/immunoblotting method, using agarose gel and visualization by an alkaline phosphatase conjugated antibody, is presented. The allele frequencies were: Hp* 1F 0. 151; Hp*1S 0.241; Hp*2FS 0.565; Hp*2SS 0.040; Hp*2FF 0.002; HpJ*0.0002. Based on these data the theoretical chance of exclusion of nonfathers in paternity cases was calculated to be 35 %. Examination of 51 families with 120 children and of 648 mother/child pairs showed no exceeptions from Mendelian inheritance. The results obtained by application of Hp subtyping to 405 paternity cases are given and the reliability of the method is discussed. A new α2‐variant occurring in the father and in two of three siblings of a Danish family is presented.
Identification and quantification of apolipoprotein D in normal human urine
Authors: Holmquist Leif 
Published: May 2005

AbstractApolipoprotein D has been identified in normal human urine, using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by immunoblotting with monospecific antibodies. Urinary apolipoprotein D appeared as a main 33 000 u protein together with a minor fraction corresponding to its partially deglycoslated species of lower molecular mass. No high molecular mass forms of apolipoprotein D naturally occurring in plasma could be detected. The apolipoprotein D mean ± SD concentration assayed with rocket immunoelectrophoresis, in urine samples from nine apparently healthy normal men, was 1.4 ± 1.0 mg/L (range: 0.2–3.0 mg/L). Among the plasma apolipoproteins, apolipoprotein D behaves uniquely as regards its excretion in urine; the other apolipoproteins belonging to the A, B, C and E groups, although of low molecular masses, are present, at most, in trace amounts in normal urine.
Resolution of rabbit liver cytochrome P450 3b and 3c (P450 IIC3 and IIIA4) from microsomal membrane by two‐dimensional gel electrophoresis
Authors: Bonfils Claude , Combalbert Jean 
Published: May 2005

AbstractTwo‐dimensional gel electrophoresis was applied to the separation of microsomal proteins and P450 isozymes. The experimental conditions of the first‐dimensional isoelectric focusing were optimized. The best results were obtained under the following conditions: (1) a mixture of two detergents (Tergitol NP 10 and CHAPS) in the focusing gel and sample buffer, (ii) a carrier ampholyte gradient between pH 6.5 and 9.0, (iii) sample loading at the anodic end of the gel, and (iv) low protein loading in the sample (below 50 μg). Four P450 forms, including P450 3b and 3c, could be resolved under these conditions.
The analysis of glycoproteins in cells and tissues by two‐dimensional polyacrylamide gel electrophoresis
Authors: Koch Gordon L. E. , Smith Michael J. 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used to identify and analyse subsets of proteins in cells and tissues. The combination of 2‐D PAGE and [125I]concanavalin A overlay revealed an extraordinary complexity and diversity in the glycoprotein profiles of different cell types. However, the glycoproteins are not expressed idiosyncratically. Rather, their expression is closely linked to the state of differentiation of a particular cell type. Such glycoproteins can therefore be used to generate antibodies specific for differentiated cells. 2‐D PAGE analyses of cellular glycoproteins also revealed a major common glycoprotein of 100 kDa. This was localised to the lumen of the endoplasmic reticulum and is referred to as endoplasmin. The combinat on of 2‐D PAGE with electroblotting and 45Ca overlay revealed that endoplasmin and several other luminal endoplasmic reticulum proteins (reticuloplasmins) are high capacity, low affinity calcium binding proteins which could function as calcium storage proteins in the endoplasmic reticulum. One of these called calreticulin is also found in the sarcoplasmic reticulum. 2‐D PAGE and 45Ca overlay has been used to demonstrate the presence of a calcium‐binding protein (CP22/sorcin) in the cytosol of rodent multidrug resistant cells. Analyses of murine serum by 2‐D PAGE revealed the presence of a novel stress protein serum amyloid P component. These studies illustrate the value of 2‐D PAGE when used in combination with detection methods which select specific subsets of proteins such as glycoproteins.
Protein synthesis initiation factor modifications during viral infections: Implications for translational control
Authors: Duncan Roger F. 
Published: May 2005

AbstractInfection of tissue culture cells with certain viruses results in the shutoff of host cell protein synthesis. We have examined virally infected cell lysates using two‐dimensional gel electrophoresis and immunoblotting to ascertain whether initiation factor protein modifications are correlated with translational repression. Moderate increases in eukaryotic initiation factor (eIF)‐2α phosphorylation are detected in reovirus‐ and adenovirus‐infected cells, as reported previously (Samuel et al., 1984; O'Malley et al., 1989). Neither vesicular stomatitis virus, vaccinia virus, frog virus III, rhinovirus, nor encephalomyocarditis virus caused significantly increased 2α phosphorylation. There were no reproducible, significant changes in eIF‐4A, eIF‐4B, or eIF‐2β in cells infected by any of these viruses. The cleavage of eIF‐4F subunit p220, such as has been previously demonstrated to occur in poliovirus (Etchison et al., 1982) and rhinovirus (Etchison and Fout, 1985), was not detected in any of the other virus infections analyzed.
A two‐dimensional gel protein database of noncultured total normal human epidermal keratinocytes: Identification of proteins strongly up‐regulated in psoriatic epidermis
Authors: Celis Julio E. , Crüger Dorthe , Kiil Jette , Dejgaard Kurt , Lauridsen Jette B. , Ratz Gitte P. , Basse Bodil , Celis Ariana , Rasmussen Hanne H. , Bauw Guy , Vandekerckhove Joel 
Published: May 2005

AbstractA two‐dimensional (2‐D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ‐SCAN and PDQUEST software) 2‐D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celis et al., Leukemia 1988, 2, 561–602) as well as by 2‐D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up‐regulated in psoriatic epidermis (Celis et al., FEBS Letters, in press).
Electrophoretic separation of nucleic acids: Evaluation by video and photographic densitometry
Authors: Freeman Steven E. , Larcom Lyndon L. , Thompson Bryan D. 
Published: May 2005

AbstractThe separation of DNA by gel electrophoresis provides a rapid method for determining size distributions of DNA in solution. Densitometric scanning of photographs of gels has been the standard method of analysis of agarose gels. However, analysis of photographs is complicated by the non‐linear response of photographic film. Charged‐coupled device video cameras have become popular for quantitative densitometry and we have used a charge‐coupled device camera to image agarose gels to quantitate DNA damage. We compare video and photographic densitometry for quantitation of ultraviolet radiation (UV)‐induced DNA damage and find that the two methods give equivalent results.
Interpretation of DNA profiles using a computerised database
Authors: Gill Peter , Werrett David J. 
Published: May 2005

AbstractAn image analyser has been utilised to store data derived from autoradiography of hypervariable loci (both multi and single locus). By examination of the electrophoretic system, it is possible to assign standard deviations to bands and then to determine whether the bands ‘match’ each other. The problems associated with the practical applications of such a system are discussed. Improvements are suggested such as the adoption of internal markers.
Preparative isoelectric focusing of reduced wheat gluten proteins
Authors: Curioni Andrea , Peruffo Angelo Dal Belin , Pogna Norberto E. 
Published: May 2005

AbstractProteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2‐mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3–10 gradient containing 50% v/v 1‐propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1‐propanol was comparable to that obtained on two‐dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.
Interaction of the vitamin D‐binding protein (groupspecific component) and its ligand 25‐hydroxyvitamin D3: Binding differences of the various genetic types disclosed by isoelectric focusing
Authors: Braun Andreas , Brandhofer Angelika , Cleve Hartwig 
Published: May 2005

AbstractThe three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin‐layer polyacrylamide gel isoelectric focusing in a pH 4.5–5.4 carrier ampholyte generated pH gradient, additionally containing N‐(2‐acetamido)‐2‐aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25‐hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points.
Improved detection of the histamine receptor on human peripheral blood mononuclear cells by crosslinking using tritiated as compared with radioiodinated histamine
Authors: Warlow Robert S. , Dempsey Susan , Carroll Raeleen , Gibson Steven , Bernard Claude C. A. 
Published: May 2005

AbstractIn order to detect histamine receptors on the surface of human peripheral blood monouclear cells, the cells were incubated in the presence of radiolabelled histamine and then the bifunctional crosslinker disuccimidyl suberate was added in various concentrations. They were then solubilized with sodium dodecyl sulphate, boiled, reduced and the lysate separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Both 3H and 125I‐radiolabelled ligands bound to a 16 kDa band, to be defined although a much clearer and obviously unequivocal signal was obtained with 3H‐labelled histamine. This molecule migrated with the same mass on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis as a 16 kDa subunit which had been purified on a histamine affinity column from Triton X‐100 solubilized mononuclear cells, indicating it to be the ligand‐binding subunit for the histamine receptor on these cells. For 3H, fluorography with EntensifyTM was required to obtain an autoradiographic signal. Although 3H took much longer to give a signal than 125I, the considerable background, artefacts and heavy lane trailing seen with [125I] histamine were completely abrogated when [3H]histamine was used. In addition, the distinction between specific and nonspecific binding was more clearly seen using [3H]histamine. The modifications reported here which improve signal detection for 3H should encourage the use of tritiated ligands in radioreceptor crosslinking, particularly those of low molecular weight which might otherwise undergo steric modification due to iodination, this having the potential for interfering with receptor ligand binding.