bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomically linked cellular protein databases derived from two‐dimensional polyacrylamide gel electrophoresis
Authors: Neidhardt Frederick C. , Appleby David B. , Sankar Pushpam , Hutton M. Elizabeth , Phillips Teresa A.
Published: May 2005

In its most useful form a cellular protein database should be genomically based, because it is the genome which determines both the total number of proteins a cell can make and the particular ones that will be made under any given condition. Such a database should trace each protein back to its structural gene, and should account for every structural gene of a cell. Recent advances in molecular biology greatly facilitate the construction of such gene‐protein databases. The mapping of genes of unidentified proteins resolved from total cell extracts on two‐dimensional gels can now be accomplished by largely biochemical methods, without the necessity of isolating mutants or performing genetic crosses. Other techniques permit one to search gels for the product of any newly discovered gene (or open reading frame) suspected of encoding a protein. Consequently, gene‐protein indices can be built independently and simultaneously from either direction – deducing the genetic map from the protein pattern, or finding the protein pattern from information encoded in the genome. A database of this sort is being constructed for the bacterium, Escherichia coli. Given the current pace of DNA nucleotide sequencing, the development of total gene‐protein indices for a variety of cells can be anticipated in the near future.
A simple, sensitive method of analyzing bacterial ribosomal DNA polymorphism
Authors: Picard‐Pasquier Nicole , Ouagued Malika , Picard Bertrand , Goullet Philippe , Krishnamoorthy Rajagopal
Published: May 2005

A rapid DNA extraction procedure and random primer labelling of Fscherichia coli ribosomal RNA with cloned reverse transcriptase have been used to establish a simple and highly sensitive method for studying ribosomal DNA polymorphism in bacteria. Examples of inter‐ and intraspecies differentiation of bacteria are given and potential applications in bacterial epidemiology and taxonomy are discussed.
Classification of mouse submaxillary gland esteroproteases by their substrate specificities
Authors: Kurosawa Nobuyuki , Ogita Zen‐Ichi
Published: May 2005

The submaxillary gland esteroproteases were separated by two‐dimensional polyacrylamide gel electrophoresis, and their súbstrate specificities were determined by histochemical staining procedures using cellulose acetate membranes. Twenty‐one proteolytically active enzymes were classified into four groups based on substrate specificities on benzoyl‐arginine‐ethylester (BAEE), N‐α‐benzoyl‐DL‐arginine‐p‐nitroanilide (BAPNA), and poly‐L‐lysine. These types were further divided into eight subgroups by their sensitivity to inhibitors and androgen dependence. These results suggest that eight groups of esteroproteases are present in the submaxillary gland of male mice.
Influence of a soluble anionic polymer on the electrophoresis of proteins
Authors: Shimura Kiyohito , Kasai Ken‐Ichi
Published: May 2005

The influence of a soluble anionic polymer on electrophoresis of proteins was studied in relation to the nonspecific ionic effect of an affinophore on application to affinophoresis. Zone electrophoresis of proteins was carried out in agarose gel in the presence of succinyl‐poly‐L‐lysine (degree of polymerization, 120) by using three electrophoresis buffers differing in ionic strength (0.06, 0.12 and 0.18) and pH (7.0 and 7.9). Proteins migrated as distinct single bands even in the presence of the polymer. The mobility of cationic proteins towards the cathode was first decreased and then increased towards the anode as the polymer concentration increased, while that of anionic proteins was not affected. The dependence of the apparent mobility changes of the proteins on the concentration of the polymer was treated quantitatively in the same way as affinity electrophoresis. The extent of the ionic interaction between a cationic protein and the polymer could be estimated as an apparent dissociation constant. It greatly depended on the ionic strength of the electrophoresis buffer. Except for the extremely cationic proteins such as lysozyme, the ionic interaction with up to 0.1 mM of the polymer could be practically suppressed by the use of 0.1 M sodium phosphate buffer (pH 7.0).
Enhanced immunodetection of blotted house dust mite protein allergens on nitrocellulose following blocking with tween 20
Authors: Tovey Euan R. , Ford Stephen A. , Baldo Brian A.
Published: May 2005

The effect of blocking nitrocellulose membranes with the nonionic detergent Tween 20 on the detection, by protein blotting, of IgE‐binding to house dust mite Dermatophagoides pteronyssinus allergens has been investigated. Tween blocking led to enhanced immunodetection of allergens despite removal of proteins from the membrane when compared to protein blocking agents which did not displace transferred components. The enhancement varied with the different mite components and, for one in particular, antigen Der p II, an increase of more than 100‐fold in IgE antibody binding occurred despite a concurrent loss of more than 90% of Der p II from the membrane. Both the enhancement of binding and loss of components from the membrane were dependent upon the time course of blocking and the concentration of Tween used.
The application of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis to the taxonomic identification of the total body protein band profiles of Diplostomum spp. metacercariae (Digenea), parasites of fish eyes
Authors: Faulkner Michael
Published: May 2005

Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis has been used to determine the metacercariae (Mc) total body protein band profiles of different Diplostomum spp. Four species of fish were investigated, roach (Rutilus rutilus) infected with D. spathaceum Mc, gywniad (Coregonus laveratus) infected with D. coregonus Mc, ruff (Gymnocephalus cernua) infected with a Diplostomum species related to D. gasterostei Mc, and perch (Perca fluviatilis) infected with D. gasterostei Mc. The four species of Diplostomum Mc were distinguished by three different bands of molecular weight, Mr 55 500, 53 500 and 52 000. A homology of polypeptide component distribution was evident for D. gasterostei Mc, from P. fluviatilis, and D. coregonus Mc, from C. laveratus, the latter showing reduced protein concentrations between bands Mr 32 000–40 000. Critical analysis of Mc polypeptide patterns showed no evidence of contamination with lens, retina or vitreous humour host eye protein. Gross morphological data for the parasites was also considered in relation to different band profiles obtained for the Diplostomum spp. Mc. Band profile analysis in conjunction with other taxonomic tests proved to be a useful tool in the identification of unknown species.
Analysis of porcine erythrocyte lysate proteins by horizontal two‐dimensional electrophoresis with immobilized pH gradient in the first dimension
Authors: Russ Ingolf , Günther Siegfried , Pirchner Franz
Published: May 2005

Horizontal two‐dimensional gel electrophoresis with immobilized pH gradients in the first dimension resulted in excellent patterns of porcine erythrocyte lysate proteins. The overwhelming excess of hemoglobin was removed by chloroform‐ethanol extraction and the number of spots further increased by acetone precipitation, revealing considerably more protein spots than detected by analyzing the untreated sample. A total of 430 protein spots have been reproducibly obtained for further analysis of genetic variations. A number of enzyme systems have been identified by visual inspection and comparison with previous results. The existence of further multiple forms for other enzymes is also apparent.
Mobility surfaces for field‐inversion gel electrophoresis of linear DNA
Authors: Crater Glenn D. , Gregg Michael R. , Holzwarth G.
Published: May 2005

The mobility of linear DNA during field‐inversion gel electrophoresis was measured as a function of molecular weight Mr, pulse time t, and field strength E. Values of Mr between 48.5 and 194 kilobase pairs (kb), E from 5 to 14 V/cm and pulse times of 0.3 to 12 s were used. The data are presented as three‐dimensional surfaces of mobility: E:t for fixed Mr or graphs of mobility: Mr:t for fixed E. The surfaces are not smoothly increasing functions of E, Mr, or t but instead show a valley with minimum mobility and a steep rise in mobility as t increases. For a field of 10 V/cm, 1 % agarose gels, and 3:1 ratio of forward: back pulse time, the forward switching time t* at which the mobility changes most rapidly is given by t* =(0.034 ± 0.003) Mr for Mr in kb and t* in seconds. The data and equations delineate the best conditions to achieve a particular separation.
Sieving of double‐stranded DNA during agarose gel electrophoresis
Authors: Serwer Philip
Published: May 2005

Agarose gel electrophoresis is used to fractionate linear, double‐stranded DNA by its length. Sieving of the gel is the cause of this fractionation and has been investigated by developing theoretical models and by quantifying sieving observed during electrophoresis. Here are reviewed the following aspects of the fractionation of linear, double‐stranded DNA by agarose gel electrophoresis: (1) the basic observations that qualitatively characterize these fractionations, (2) evidence for the deformation of DNA's random coil, (3) quantitative analysis of the relationship of observed electro‐phoretic mobility to the DNA's length, (4) theoretical models that have been developed to explain data presented in Sections 1–3, (5) observations not yet quantitatively explained by models, and (6) some aspects of the use of a variable electrical field (pulsed‐field gel electrophoresis) to improve separations.
Measurement of protein‐DNA interaction parameters by electrophoresis mobility shift assay
Authors: Fried Michael G.
Published: May 2005

Native gel electrophoresis (mobility shift) assays may be used to obtain quantitative information about the site distribution, equilibria and kinetics of protein‐DNA interactions. These applications depend on the ability of the electrophoretic system to resolve the reaction components, and on their stabilities during the separation process Factors which affect the lifetimes and mobilities of protein‐DNA complexes during electrophoresis include reaction and electrophoresis buffer composition, pH, and ionic strength; the presence of low molecular weight effectors and enzymatic substrates; the nature and concentration of the gel matrix; the temperature; the molecular weights of protein and DNA; the stoichiometric ratios of their complexes; and the possibility of conformational and configurational isomerization of reaction components. We discuss how these factors influence the acquisition of quantitative data from electrophoretic patterns and band intensities, and present formulas for the estimation of equilibrium constants and rate constants for prototypical DNA‐protein interactions.
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

Chromosome‐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

This 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

A 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

The 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

Group 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

Isoelectric 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

For 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

A 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

A 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

Commercial 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

In 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

Formulations 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

Haptoglobin 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

Apolipoprotein 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

Two‐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

Two‐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

Infection 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

A 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

The 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

An 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

Proteins 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

The 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

In 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.
Sequence analysis of proteins separated by polyacrylamide gel electrophoresis: Towards an integrated protein database
Authors: Aebersold Ruedi , Leavitt John
Published: May 2005

Improved technologies or the synergistic use of complementary methods enhance the efficiency of research and permit the exploration of new approaches for the investigation of complex problems. High sensitivity protein sequence analysis and polyacrylamide gel electrophoresis are such complementary methods. Here we summarize the current status of high sensitivity sequence analysis of proteins separated in polyacrylamide gels and discuss strategies by which this technology can cnhance biological research by generating new approaches for the solution of complex, multifacetted problems. Finally, we outline lmminent technological advances in the area of high sensitivity protein sequence analysis and argue that further technological developments will ultimately lead to the generation of an integrated protein database (containing structural and functional as well as physiological information in an easily accessible form) of all the proteins separated by high resolution two‐dimensional gel electrophoresis.
Application of micellar electrokinetic chromatography to pharmaceutical analysis
Authors: Nishi Hiroyuki , Terabe Shigeru
Published: May 2005

Electrokinetic chromatography is a new type of analytical separation method which belongs to the group of high performance capillary electrophoretic techniques but whose separation principle is based on that of chromatography. The solute distributes itself between a carrier and the surrounding medium. The carrier, which corresponds to the stationary phase in conventional chromatography, can be transported by electrophoresis with a different velocity from the surrounding medium. The separation is achieved by the differential solute distribution and the differential migration of the carrier. The charged molecules or charged molecular aggregates are employed as the carrier. Various kinds of carriers are available for electrokinetic chromatography along with different partition mechanisms. Among them, micellar electrokinetic chromatography, which employs an ionic micelle as a carrier, has become the most popular method because of its unique and attractive characteristics as well as the separating capability of electrically neutral or nonionic solutes in comparison with capillary zone electrophoresis. The present paper describes the principle, separation characteristics and its application to the analysis of pharmaceuticals.
The separation and detection of alkaline oligoclonal IgG bands in cerebrospinal fluid using immobilised pH gradients
Authors: Cowdrey Geoffrey , Gould Barry , Rees John , Firth Gary
Published: May 2005

A method is described for the separation and detection of highly alkaline IgG bands in unconcentrated cerebrospinal fluid (CSF). These bands are frequently found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system, particularly in the case of multiple sclerosis, and their detection is an important aid in clinical diagnosis. An isoelectric focusing technique using an immobilised pH gradient in polyacrylamide gel has been developed over the pH range 7–10, producing a linear and stable pH gradient with excellent resolution. After electrofocusing, the protein patterns were blotted onto polyvinylidene difluoride membranes and visualised using anti‐human IgG followed by an enzyme‐labelled second antibody. Blotting could be carried out by capillary diffusion for up to 16 h duration without any loss in resolution. Using this method, highly alkaline intrathecal IgG bands were found in the cerebrospinal fluid of all of the 14 multiple sclerosis patients. There were also 2 patients with alkaline IgG bands in their cerebrospinal fluid who were not diagnosed as multiple sclerosis. By contrast, no alkaline IgG bands with an isoelectric point (pI) greater than 8.6 were found in any of the serum samples studied (n = 50) from patients with various neurological disorders including multiple sclerosis.
Inbred testing of tomato (Lycopersicon esculentum L.) F1 varieties by ultrathin‐layer isoelectric focusing of seed protein
Authors: Van Den Berg Bartel M.
Published: May 2005

To test seed lots of tomato F1 hybrid varieties for the presence of undesirable inbred seeds by electrophoresis, a method has been developed on the basis of ultrathin‐layer isoelectric focusing. The method is based on the genetic variation of the seed protein PRS‐1 which could be visualized by isoelectric focusing of a 5 mm NaCl‐soluble seed protein extract in a pH 6–9 gel followed by protein staining. Two genetic variants of the PRS‐1 protein, PRS‐1+ and PRS‐11, were found among open‐pollinated varieties, as well as among F1 hybrid varieties. The isoelectric points (pI) of the PRS‐1 proteins are 7.1 and 6.1 for PRS‐1+ and PRS‐11, respectively. The PRS‐1 protein is unique to seed tissue and is located primarily in the embryo. A genetic 1:2:1 segregation of the gene Prs‐1 among several F2 populations shows monogenic inheritance. Analysis of commercial F1 hybrid varieties from several seed companies indicated that the Prs‐11 allele, in contrast to the Prs‐1+ allele, is primarily present in gene pools of „Moneymaker type”︁ tomatoes. The described method is generally applicable to all tomato F1 varieties that are heterozygous for the gene Prs‐1. With the described method one person can routinely analyze more than 768 seeds per day.
Preparative free‐flow isoelectric focusing: Modeling and experiments
Authors: Poux Martine , Bertrand Joël
Published: May 2005

Free‐flow isoelectric focusing was adapted to preparative scale separations and chemical engineering methods were used to describe the main mechanisms operating in the apparatus. A mixture of human serum albumin (pI 4.6) and β‐lactoglobulin (pI 5.22) was separated in pH gradients, generated with carrier ampholytes of different origin and covering the pH ranges 4‐6.5, 3.5‐5, 4‐5.5 and 4.5‐5.0. Best results were obtained in the pH 4‐5.5 range. The experimental results have validated the results obtained with a numerical model.
Free‐flow electrophoresis for the purification of proteins: II. Isoelectric focusing and field step electrophoresis
Authors: Kuhn Reinhard , Hoffstetter‐Kuhn Sabrina , Wagner Horst
Published: May 2005

Two modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of α‐amylase from an E. coli protein extract. Furthermore the principle of field step electrophoresis is presented. The most important factors influencing the efficiency: (i) residence time, (ii) conductivity of the sample and (iii) sample zone width, are discussed. The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling‐up field step electrophoresis. This approach permits a throughput of about 20 mL sample solution per minute.
Separation of antibiotics by autofocusing
Authors: Sova Otto
Published: May 2005

The autofocusing separation of nine antibiotics was investigated: tetracycline and chloramphenicol in large‐scale batches, and seven others in gram preparation amounts. All except one were found to be hetergeneous in autofocusing, and 2‐4 well‐characterized fractions emerged. These heterogeneities are described and the industrial utilization of the procedure is suggested.
Quantitative exploration of the REF52 protein database: Cluster analysis reveals the major protein expression profiles in responses to growth regulation, serum stimulation, and viral transformation
Authors: Garrels James I. , Franza B. Robert , Chang Cecile , Latter Gerald
Published: May 2005

Quantitative protein databases reveal the response of cells to experimental variables, such as exposure to growth factors or transfection with a transforming gene. The nature of the response depends on the type of cell and its internal state at the time of the stimulus. By constructing a protein database to study a given cell line, we can better understand the differentiated state of the cell, the growth regulatory mechanisms it employs, the particular mechanisms it uses to cope with its environment, and the ways these mechanisms may have been compromised through mutation or transformation. The REF52 database is a quantitative database designed to study growth control and transformation in a well‐defined family of normal and transformed rat cell lines. The database, which has been described and analyzed elsewhere (J. I. Garrels and B. R. Franza, J. Biol. Chem. 1989, 264, 5283–5298 and J. I. Garrels and B. R. Franza, J. Biol. Chem. 1989, 264, 5299–5312) is further explored here using cluster analysis. This method reveals the most common protein expression profiles for each series of two‐dimensional gels without requiring any prior hypothesis or queries on the part of the investigator. This study reveals, for each experiment, large and small clusters of protein expression profiles, most of which have readily apparent biological meaning. For example, large clusters of proteins induced or repressed during growth to confluence have been revealed, and several clusters of transformation‐sensitive proteins reveal differential effects of transformation by DNA‐ and RNA‐tumor viruses. This analysis extends our earlier quantitative explorations of the REF52 protein database and helps to show how such a database can be used to provide context and guidance for molecular studies of regulation in a given cell system.
Automated amplification and sequencing of human mitochondrial DNA
Authors: Sullivan Kevin M. , Hopgood Romellé , Lang Brian , Gill Peter
Published: May 2005

Part of the human mitochondrial D‐loop region was amplified by two successive rounds of polymerase chain reaction (PCR) amplification. In the second PCR reaction, nested primers were used, of which one contained the M 13–21 universal primer sequence. By using nonequal concentrations of primers in the second amplification, single‐stranded DNA was generated. This was then sequenced directly by the dideoxy chain termination method using dye‐labelled universal sequencing primers in conjunction with a fluorescence‐based DNA sequencer. This enabled a 403‐base‐pair hypervariable segment of the D‐loop region to be readily sequenced in a single reaction. This paper describes a protocol which enables mitochondrial sequence information to be generated rapidly and automatically. It is likely to be of importance in forensic analysis where the DNA is too degraded or of insufficient quantity to be analysed by other techniques.
Structural analysis of recombinant protein prepared by semi‐dry electroblotting after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Fausset Patricia R. , Lu Hsieng S.
Published: May 2005

Proteins that were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were electroblotted onto polyvinyliden difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N‐terminal sequence analysis. A semi‐dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N‐Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N‐terminal sequence analysis of the purified electroblotted samples. Consequently, time‐consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.
Computerized methods for analyzing two‐dimensional agarose gel electropherograms
Authors: Aldroubi Akram , Unser Michael , Tietz Dietmar , Trus Benes
Published: May 2005

Previous methods interpret zonal or polydisperse gel patterns of two‐dimensional Serwer‐type gels in terms of size and free mobility (surface net charge density). These two parameters have been determined for each component without quantitatively measuring the abundance of the components. The present study advances these previous methods by determining the relative concentration of each component by computer evaluation of densitometrically analyzed gel patterns. Suitable procedures and their underlying algorithms are presented. The mathematical routines are implemented in a user‐friendly software package, called GelFit and designed for a Macintosh personal computer. The program input sonsists of digitized images of gel staining patterns exemplified by those obtained from electrophoresis of native sub‐cellular‐sized particles. The data are processed through the following steps: (i) Noise reduction and calibration. (ii) Geometrical transformation of the pattern onto a rectangular size/free mobility coordinate system using rationales of the extended Ogston model. (iii) Analysis of the transformed image to determine density maxima, density profiles along iso‐free‐mobility or iso‐size lines, curve fitting of one‐dimensional profiles or two‐dimensional surfaces using Gaussian functions and curve stripping of surfaces to determine the possible number of particle populations.