bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of convex Ferguson plots in agarose gel electrophoresis by empirical computer modeling
Authors: Tietz Dietmar , Chrambach Andreas
Published: May 2005

Agarose gel electrophoresis has been shown to give rise to non‐linear plots of log (mobility) vs. gel concentration of spherical viruses (Serwer,[1]) and cellular vesicles (Gottlieb et al.[2]). This finding also applies to proteins as shown in this study. Considering that in the non‐linear plot, the slope becomes a function of gel concentration, it is possible to determine particle properties and gel parameters by a modification of the conventional method derived from the Ogston theory for long‐fiber gels. This treatment shows: (a) In application to data obtained from gel electrophoresis (0.4 to 1.6 % agarose) of viruses (13 to 42 nm radius) and with increasing gel concentration: (i) an increase of apparent total fiber length per g agarose matrix, (ii) a reduction of apparent fiber radius and (iii) a constant fiber volume (per g matrix material) of the agarose fiber. The values of the fiber radii identify the fibers as agarose supercoils (or aggregates of it) with a radius of 20–55 nm. (b) In application to data obtained from gel electrophoresis (1.2 to 8 % agarose) of proteins (1.7 to 5.8 nm radius): a fiber volume indicative of additional sieving by the agarose double helix (of known radius of 0.5–2 nm). This is in agreement with a previous suggestion by Serwer [1] that proteins are able to penetrate into the double‐helical network of which the agarose supercoil consists. (c) The likelihood of a continuous transition from case (a) to case (b).
Avoiding liquid exudation on the surface of rehydrated gels used for hybrid isoelectric focusing in carrier ampholyte supplemented immobilized pH gradients
Authors: Altland Klaus , Hackler Rolf , Rossmann Ute
Published: May 2005

Recently, we described hybrid isoelectric focusing in rehydrated polyacrylamide gels as an analytical procedure which is based on separation in immobilized pH gradients supplemented with low concentrations of carrier ampholytes (Altland and Rossmann, Electrophoresis 1985, 6, 314–325). When using this procedure at various pH ranges, in the presence of reducing agents and carrier ampholytes at different concentrations and from different suppliers, over prolonged time intervals for separation and at various acrylamide concentrations in the gel, liquid exudation has been observed on the gel surface which may result in a confluent liquid layer adversely affecting the final separation pattern. The effect is demonstrated and procedures are described to overcome the phenomenon by adding polyols like glycerol, sorbitol, sucrose or dextrans to the rehydration solution. Comparable effects are achieved when these components are added at the same concentration by weight rather than by moles. The addition of 10 g % Dextran 8 or 20 g % sucrose or sorbitol to the rehydration solution prevents liquid exudation for several hours. These additives are compatible with high concentrations of urea, with reducing agents and with neutral detergent (Triton X‐100).
A technical note on esterase D subtyping
Authors: Rocha Jorge , Amorim AntóNio
Published: May 2005

The electrophoretic behavior of esterase D (E.C. 3.1.1.1.) allozymes 1, 2 and 5 was studied in various buffers and pH ranges, and compared with results obtained on isoelectric focusing. It is shown that the relative mobility of ESD 2 and 5 allozymes is reversed according to the pH of the buffer system. In acidic pH ranges the mobility is: cathode 1<2<5 anode; using the same buffer system in basic pH ranges the following order is observed: 1<5<2. Technical improvements in agarose gel electrophoresis leading to a considerable economical advantage over the previously described methods are presented.
On the relationship of amino acid composition to silver staining of proteins in electrphoresis gels
Authors: Gersten Douglas M. , Wolf Pamela H. , Ledley Robert S. , Rodriguez Lewis V. , Zapolski Edward J.
Published: May 2005

Development of techniques for silver staining of proteins separated by polyacrylamide gel electrophoresis has increased experimental detection limits. However, the precise basis for the reaction between silver and polypeptides is still unclear and, depending upon the choice of silver reagent, may even differ. We compared protein stain intensity with silver diamine (ammoniacal silver) and silver nitrate reagents based on amino acid composition by determining the protein amount required to generate an arbitrary staining intensity and then calculating moles of each amino acid present in some representative proteins. We compared these values for each amino acid, taken singly, as well as for combinations of two and more. In no case could staining be attributed entirely to specific or classes of amino acids, thus supporting the argument that an inherent difference exists between primary reactive centers for the two staining reagents.
Decrease in the mobility of proteins during electrophoresis in linear gradients of polyacrylamide
Authors: Lambin Patrick , Herance Nicole , Fine Jean‐Marius
Published: May 2005

Proteins with molecular weight between 66 000 and 950 000 were subjected to electrophoresis in 3 to 30 % linear gradients of polyacrylamide with volt‐hour products between 2000 and 20 000. From the experimental data it was concluded that displacement of proteins decreased proportionally to the logarithm of time of electrophoresis. With these coordinates the fitness of the curves was found to be excellent (correlation coefficients were better than 0.993 for the analyzed proteins). These results support the hypothesis that there is no limit to be reached by a protein during its migration in a linear gradient of polyacrylamide. In addition we observed that the rate of decrease in mobility was proportional to the size of the protein.
Human antibody‐secreting cells enriched by free flow electrophoresis
Authors: Bauer Johann , Hannig Kurt
Published: May 2005

Mononuclear leukocytes of the human peripheral blood were separated by counter‐current centrifugal elutriation. In this way a group of lymphocytes was isolated, which contained B‐lymphocytes with an electrophoretic mobility (EM) of 0.9 × 10−4 (cm2 V−1 s−1) and T‐lymphocytes with an EM of 1.1 × 10−4 (cm2 V−1 s−1). This group of lymphocytes was incubated for 7 days in the presence of monocytes and pokeweed mitogen. Then the culture supernatants were screened. Non‐specific human antibodies were found in all culture supernatants. In 20% of the supernatants human antibodies against sheep red blood cells could be detected. The viable cells of the cultures were separated into 5 fractions by free flow electrophoresis. Then the cells of the different fractions were tested for their capability to produce antibodies. The tests revealed that antibody‐secreting cells had an EM around 0.9 × 10−4 (cm2 V−1 s−1) and that the greater part of the cells, isolated from pokeweed mitogen cultures, had a significant higher EM. Thus free flow electrophoresis could be applied to enrich antibody‐secreting cells ninefold.
Isotachophoretic focusing on thin gel slab: A new and powerful electrophoretic method of protein analysis
Authors: Charlionet Roland , Bringard Aline , Davrinche Christian , Fontaine Marc
Published: May 2005

The theoretical principle and experimental verification of a new separation method for proteins in thin gel slabs called “isotachophoretic focusing” are presented. Isotachophoretic focusing is basically isotachophoresis with two new features: (i) By using adequate spacers a continuous mobility spectrum is generated. Under isotachophoresis conditions, these spacers give rise to a linear electric field gradient in which proteins can be separated in form of focusing bands. The synthesis and isolation of suitable spacers for isotachophoretic focusing are described. (ii) The stack is immobilized or, at least, its migration is retarded by exploiting the counterflow of electroendosmosis resulting from the action of the electric field on charged groups linked to the gel. Immobilization of the stack allows its formation to equilibrium and the protein separation process to continue as long as necessary. Details are given concerning the experimental set‐up and factors involved in the optimization of the technique. The separations obtained by isotachophoretic focusing are compared with those obtained by classical electrophoresis.
Transfer of proteins from plastic‐backed isoelectric focusing gels to nitrocellulose paper
Authors: Albaugh George P. , Chandra G. Ram , Bottino Paul J.
Published: May 2005

Proteins were transferred to nitrocellulose paper from plastic‐backed isoelectric focusing gels. The technique involves a two‐step blotting procedure. In the first step, simple press blotting was used to establish adhesion between the gel and overlaying nitrocellulose paper. This step facilitated the removal of the plastic backing. In the second step, electroblotting produced a pair of bi‐directional replica blots. Incubation of the gel‐nitrocellulose paper sandwich in buffers containing T ween 20 permitted the intact release of the gel from the blots. The resulting blots were successfully stained with India ink or probed with antiserum.
Enzyme polymorphism in bacteria: Study of molecular relatedness by combined isoelectric focusing‐electrophoresis
Authors: Picard Bertrand , Krishnamoorthy Rajagopal , Goullet Philippe
Published: May 2005

The molecular relationships between electrophoretic variants of different bacteria, defined in two‐dimensional electrophoretic profiles of acid phosphatase, esterases, glutamate dehydrogenase and malate dehydrogenases, were analyzed by combined isoelectric focusing‐electrophoresis. The mobility curves of enzymes confirmed the monomorphism of variants showing identical isoelectric point (pI) and relative electrophoretic mobility (MF) values and refined the polymorphism analysis of variants showing distinct pI and/or MF values.
Electrophoretic variability in human head hair: Polyacrylamide gel electrophoresis of hair proteins in the presence of sodium dodecyl sulfate and urea
Authors: Gerhard Michael
Published: May 2005

A fast and simple procedure for routine typing of human head hair is described. Hair proteins, extracted in a solution containing sodium dodecyl sulfate, urea and dithiothreitol were separated electrophoretically in the presence of 6 M urea. On examination of hair from 445 different individuals, eight characteristic polypeptide patterns (phenotypes) could be distinguished and were arbitrarily named K1 to K8. Three of them, K1, K2 and K3 occur frequently, especially K1, which is found in approximately 79% of the individuals investigated. The eight phenotypes are characterized by the different number and patterns of major polypeptide bands in the range of 45 to 60 kDa. Based on the high reproducibility it can be assumed that a given phenotype is specific for the head hair of an individual. Electrophoretic keratin typing is a promising tool for hair analysis in genetic and forensic investigations.
A comparison of some adjustment techniques for use with quantitative spot data from two‐dimensional gels
Authors: Kuick Rork D. , Hanash Samir M. , Chu Ernest H. Y. , Strahler John R.
Published: May 2005

Quantitative analysis of polypeptide differences among sets of identically prepared two‐dimensional polyacrylamide gels is facilitated by the use of mathematical transformations of the spot integrated intensity data for each gel in order to make comparisons. Such adjustments are needed because technical factors cause the stained polypeptide spots displayed on a particular gel to simultaneously appear darker or lighter than on another gel. We have compared three different adjustment schemes by applying each to the same set of data and testing to determine which gives smaller coefficients of variation for a large battery of spots. The adjustment that gave the best results is one based on regressing the spot integrated intensities of a standard gel versus each “target” gel and using the fitted line to transform the spot integrated intensities on the target gel. This adjustment is intuitive, easy to compute, and performed well regardless of whether the spots with large variability were included in the data or not.
Polystyrene latex particles as size standards in quantitative agarose gel electrophoresis: Application to three plant viruses
Authors: Gombocz Erich , Tietz Dietmar , Hurtt Suzanne S. , Chrambach Andreas
Published: May 2005

Negatively charged polystyrene latex particles with electron microscopically determined radii of 22.4, 26.9, 27.6, 43.5, 44.2 and 59.4 nm (nominal radii provided by the manufacturer of 28.5 to 75 nm) were found to be applicable as size standards for the quantitative agarose gel electrophoresis of viruses and cellular particles in that size range. In electrophoresis at pH 6.5, 0.03 M ionic strength in presence of 10 mM 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate (CHAPS), the polystyrene particles exhibit non‐linear, reproducibly convex‐sigmoidal Ferguson plots. This curve shape is compatible with a model specifying a dependence of gel fiber dimensions and of particle size on the gel concentration. Based on a standardization of the agarose fiber by the polystyrene latex particles, computer simulation methods were used to determine the effective radii (without added Ca++) of turnip crinkle virus (TCV), hibiscus chlorotic ringspot virus (HCRSV) and pelargonium flowerbreak virus (PFBV) as 29.4, 24.2 and 22.1 nm, and their net charges as free mobilities, μo of 9.89, 10.87 and 16.20 · 10−5 cm2/s/V, respectively. Within the size range of the standards, a simplified procedure for determination of particle size from curved Ferguson plots appears applicable as a first approximation (Zwaan method). When electrophoresis was conducted in the presence of 5 mM Ca++, the effective particle radii of TCV, HCRSV and PFBV were reduced to 12.8, 20.3 and 18.3 nm, respectively. This compares with previous electron microscopic estimates of approximately 15 nm for TCV, HCRSV and PFBV. The observed increase of effective particle radii in the absence of Ca++ corroborates the particle swelling and/or shape change at pH's ≥ 5.5 in the absence of Ca++ which is known to occur with other plant viruses.
Protein precipitation induced by alkaline Immobilines for isoelectric focusing in immobilized pH gradients: Causes and remedies
Authors: Rabilloud Thierry , Gelfi Cecilia , Bossi Maria Luisa , Righetti Pier Giorgio
Published: May 2005

The mechanism by which three basic Immobilines (pK's 6.2, 7.0 and 9.3) precipitate proteins in solution seems to be due to the presence in these three monomers of variable amounts of oligomers, spontaneously formed during storage by polymerization. These polymerization products range in size from short oligomers (dimers and trimers) to polymers with a molecular mass more than 2000 Da. Oligomers are present in all batches of precipitating Immobilines in substantial amounts, usually at least 15–20%. Only the pK 8.5 Immobiline, in general unable to precipitate proteins from solution, has been found to contain low levèls of oligomers, typically less than 3%, possibly due to intrinsic stability against polymerization, or to its ability to spontaneously hydrolyze in water solutions. None of the batches of pK 6.2, 7.0 and 9.3 Immobilines, unable to precipitate proteins in solution, was found to contain polymers but exhibited a bimodal elution peak from a Bio‐Gel P‐2 sieve gel chromatography, probably corresponding to dimers and trimers. Upon mild polymerization (highly diluted persulfate in presence of oxygen) all these batches were found to precipitate proteins and to contain higher oligomers and polymers. By testing column eluates, it was found that the precipitation power begins at the level of polymers with 10–12 residues, suggesting that these relatively short chains already possess a strong cooperativity in their chemical behavior in solution. Work is in progress to find a suitable solution for completely inhibiting two of the most noxious reactions occurring to Immobilines: hydrolysis and polymerization. There still remains a third problem, which will require a longer‐term investigation: the intrinsic hydrophobicity of the four basic Immobilines. The basic Immobiline homopolymers present two unlike groups for interaction: ionic on one side, hydrophobic on the opposite end. Acidic Immobilines, even when reacted to form much larger polymers, are unable to precipitate ferritin from solution, suggesting that their strongly hydrophilic surface suppresses one of the two binding regions. In a similar manner, when batches of pK 7.0 Immobiline, able to precipitate ferritin, where rendered more hydrophilic by methylolation, their protein precipitation power was abolished, even, though the polymers had not been eliminated. While waiting for the synthesis of more ‘hydrophilic’ Immobilines, a partial remedy is to admix carrier ampholytes (CA) to Immobiline gels. The CA's act as shielding ions, minimizing hydrophobic interactions among proteins and the Immobiline matrix. Mixed CA‐IEF gels should never be prefocused, as the shielding action of CA is maximal in the unfocused state.
A micromethod for typing haptoglobin in bloodstains
Authors: Dimo‐Simonin Noëlle , Brandt‐Casadevall Conxita , Gujer Hans‐Rudolf
Published: May 2005

A micromethod of discontinuous polyacrylamide gel electrophoresis is described for phenotyping human haptoglobin in bloodstains. Pretreatment of samples is not required. This procedure is easy, fast and can detect haptoglobin phenotypes in 0.02 μL blood in experimentally produced stains.
A tetrazolium method for peroxidase staining: Application to the antibody‐affinity blotting of α‐fetoprotein separated by lectin affinity electrophoresis
Authors: Taketa Kazuhisa
Published: May 2005

A sensitive staining method of peroxidase labels on nitrocellulose membrane was developed with phenol as an enzyme substrate and NADH as a reducing agent for Nitro Blue tetrazolium to form formazan. The tetrazolium method for peroxidase staining, when applied to the antibody‐affinity blotting of α‐fetoprotein (AFP) after agarose gel electrophoresis, allowed us to detect as little as 3 ng/mL AFP with 4 μL of sample volume, covering a wider linear range of color production up to 200 ng/mL AFP as compared with the conventional diaminobenzidine stain. Accordingly, the proportion of intensities of AFP bands separated by lectin affinity electrophoresis could be determined without employing a calibration curve for each assay.
Subtyping of factor B by agarose gel electrophoresis
Authors: Suzuki Koichi , Harumoto Terue , Ito Shigenori , Matsumoto Hideo
Published: May 2005

Conventional agarose gel electrophoresis (AGE) in Tris/glycine/barbital buffer (TGVB) could classify the common allotype “F” of factor B (BF) into two kinds of subtypes (Fb1 and F). Analysis of mother‐child pairs in paternity cases confirmed inheritance of these suballeles in the same manner as other alleles at the BF locus. The gene frequencies were estimated at 0.0154 for BF*Fb 1 and 0.1492 for BF*F through the study of 325 unrelated individuals. After treatment of serum samples with zymosan, the polymorphic band of BFFb 1 appeared in the Ba fragment, migrating between those of BFF and BFS. A technique of immunoblotting could demonstrate the BF patterns after AGE. This study provides a simple method for the detection of the BF subtypes and their electrophoretic characterization.
High resolution two‐dimensional electrophoresis of the proteins and macromolecular constituents of beer and wine
Authors: Marshall Thomas , Williams Katherine M.
Published: May 2005

The proteins/macromolecular constituents of unconcentrated/unfractionated beer and wine (50–75 μL) have been analyzed by high resolution two‐dimensional electrophoresis and silver staining. The beer patterns are dominated by up to four horizontal strings of spots of Mr 34 000–43 000 and pI 5.0–7.0 which have been tentatively identified as proteins previously isolated in association with foam and haze formation. Reproducible qualitative and quantitative differences were detected in different commercial beers. Distinctive patterns were also obtained with the wines.
Adsorption, immunoadsorption and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of proteins using nitrocellulose membrane filters
Authors: Luescher Immanuel F.
Published: May 2005

A rapid analytical procedure is described to perform sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of proteins specifically adsorbed on antibody‐coupled nitrocellulose membrane filters. Purified antibodies were adsorbed on nitrocellulose filters and were immobilized by treatment with dilute glutaraldehyde. Efficient adsorption of specific antigen occurred upon filtration of the antigen solution (e. g. cell supernatant or cell lysate) within a few minutes. Theim‐munoadsorbed proteins were released from the membrane filters in the presence of urea, Nonidet P‐40 detergent and electrophoresis sample buffer and were directly analyzed by SDS‐PAGE. All adsorptions and washing steps were performed by filtration utilizing a filtration manifold. This allowed the processing of a large number of samples in a short time. Furthermore, and in contrast to the Western blotting method, antigen‐antibody recognition preceeded gel electrophoresis, which greatly reduced potential failures in antigen recognition. Similarly, SDS‐PAGE of proteins non‐specifically adsorbed on nitrocellulose filters was also possible. Dilute protein solutions, like cell supernatants, were filtered through nitrocellulose membrane filters. Hereby proteins, but not low molecular weight compounds such as inorganic salts, amino acids or detergents, were rapidly and quantitatively adsorbed. The adsorbed proteins were released from the filters and subjected to SDS‐PAGE as in the previous method.
Differentiation between Haemophilus paraphrophilus, H. aphrophilus, H. influenzae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, P. haemolytica, and P. ureae by high resolution two‐dimensional protein electrophoresis
Authors: Olsen Ingar , Rosseland Sissel K. , Thorsrud Anne K. , Jellum Egil
Published: May 2005

The cellular proteins of Haemophilus paraphrophilus (ATCC 29242, ATCC 29241T, ATCC 29240, HK 477, HK 319, HK 159), H. aphrophilus (ATCC 33389T), H. influenzae (ATCC 31441), Actinobacillus actinomycetemcomitans (ATCC 33384T), Pasteurella multocida (NCTC 10322T), P. haemolytica (9380T), and P. ureae (NCTC 10219T) were analyzed by high resolution two‐dimensional protein electrophoresis and silver staining. The electrophoretic protein patterns of Haemophilus, Actinobacillus and Pasteurella were quite distinct. Also the patterns of P. multocida, P. haemolytica and P. ureae differed markedly. Except for H. paraphrophilus ATCC 29242, it was difficult to distinguish H. paraphrophilus from H. aphrophilus strains. Excluding ATCC 29242, H. paraphrophilus strains were quite similar. A. actinomycetemcomitans could easily be distinguished from all species tested, including the related H. aphrophilus, H. paraphrophilus, P. haemolytica, and P. ureae. High resolution two‐dimensional protein electrophoresis is likely to contribute to a more unifying concept of bacterial species. Among the organisms examined only H. aphrophilus and H. paraphrophilus seem to requires revision of their current taxonomic positions.
Effect of sialic acid on the electrophoretic mobility of mouse splenic lymphocytes
Authors: Shimizu Motomu , Iwaguchi Takao
Published: May 2005

The effect of sialic acid on the electrophoretic mobility (surface charge) of mouse splenic lymphocytes was investigated. On treatment of whole spleen and T cells with neuraminidase, their electrophoretic mobilities decreased to one‐half of the initial values. Calculation of the surface charge from the electrophoretic mobility of lymphocytes before and after treatment with the enzyme indicated that one‐half of the negative surface charge, which determines the electrophoretic mobility, was due to neuraminidase‐sensitive sialic acid in mouse lymphocytes. However, the calculated decrease in the surface charge did not coincide with the amount of sialic acid released from the cell membrane on the enzyme treatment of lymphocytes.
Identification of bovine fetal and adult serum/plasma proteins by two‐dimensional electrophoresis and immunochemical staining
Authors: Manabe Takashi , Takahashi Yuko , Okuyama Tsuneo
Published: May 2005

Bovine fetal and adult serum/plasma proteins were analyzed by two‐dimensional electrophoresis in the absence of denaturants. Bovine serum proteins were identified by electrophoretic transfer from gel slabs followed by immunochemical staining. Amine oxidase was identified by activity staining. Antisera against human plasma proteins were employed for the immunochemical identification, together with antisera to bovine proteins. Developmental changes of fetal serum proteins including α‐fetoprotein were visualized.
Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of human salivary proteins including pink‐violet components: Range, distribution and response to 2‐mercaptoethanol
Authors: Marshall Thomas , Williams Katherine M.
Published: May 2005

Mid‐morning specimens of unstimulated whole saliva were collected from 160 apparently healthy individuals and analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis following sample preparation with or without 2‐mercaptoethanol. Coomassie Brilliant Blue R‐250 staining revealed blue and pink‐violet salivary protein components. The latter, detected in approximately 60 % of the individuals, formed a limited range of patterns combining 2–8 of 11 diffuse zones of Mr 10 000–100 000. The pink‐violet components were unaffected by 2‐mercaptoethanol, urea or prior centrifugation of the sample and were not invariably present or absent in repeat samples from the same individual. The patterns of the blue staining components were more reproducible for the purpose of human individualisation but were affected by 2‐mercaptoethanol or prior centrifugation of the sample. The patterns of both the blue and pink‐violet components were not affected by sample storage at −70 °C. Only 5 μL of saliva were required for analysis giving excellent resolution over the Mr range 10 000–500 000.
Separation accuracy of free flow electrophoresis as proved by flow cytometry
Authors: Bauer Johann , Kachel Volker
Published: May 2005

The separation accuracy of the free flow electrophoresis ACE 710 device was proved by immunological methods. Several cell populations of human peripheral blood were purified using physical cell isolation methods such as countercurrent centrifugal elutriation and cell electrophoresis. The purified cell fractions were treated with fluoresceinisothiocyanate‐labelled antibodies. The percentages of cells in each fraction binding the antibodies of interest were determined by flow cytometry. The analyses revealed that with the help of free flow electrophoresis, given cell population of human peripheral blood can be highly enriched and that preenrichment of minor cell populations enhances the efficacy of a flow cytometer.
Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels
Authors: Strahler John R. , Hanash Samir M. , Somerlot Luke , Bjellqvist Bengt , Görg Angelika
Published: May 2005

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding ∼ 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion‐containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1–2 mM Tris base, or at the anode for gels rehydrated with 1–2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris‐containing gels, or at the cathode, for acetate‐containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading. Salt concentrations in excess of ∼ 0.15 M in the sample result in electroosmotic removal of water from the sample before the proteins can enter the gel, with resultant precipitation of the sample at the application site. Local pH extremes can be created at the application point, which may produce delayed sample entry or protein precipitation.
Immunostaining of rocket immunoelectrophoresis precipitates too weak for identification following staining with Coomassie Brilliant Blue
Authors: Hansen Svend A.
Published: May 2005

Low enzyme concentrations can be determined quantitatively using rocket immunoelectrophoresis if the resolved peaks are transferred electrophoretically to a nitrocellulose membrane and immunostained.
Physical identification of a virus in a crude leaf extract by its ferguson plot in agarose gel electrophoresis
Authors: Orbán László , Chrambach Andreas
Published: May 2005

Crude extracts of turnip crinkle virus upon agarose gel electrophoresis yield (i) virus patterns unperturbed by contaminants; (ii) plots of mobility vs. gel concentration (Ferguson plots) parallel with those of the purified virus. The parallelism suggests similarity in size and shape but a lower net charge for the crude virus. This result is obtained when gel electrophoresis is carried out either in a continuous buffer or in a discontinuous (moving boundary electrophoresis) buffer system. The latter mode has the substantial benefit of electrophoretic (auto‐)concentration of dilute virus sample prior to resolution. Thus, the Ferguson plot analysis in a discontinuous buffer system of turnip crinkle virus can be viewed as a model procedure for the physical identification of other viruses contained in dilute extracts, feasible even in the absence of a prior knowledge as to the nature of, or isolation of, the virus.
Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients
Authors: Knisley Keith A. , Rodkey L. Scott
Published: May 2005

Serum was collected from rabbits at 2‐day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific anti‐tetanus or anti‐micrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.
Isoenzyme analysis of lichen algae in immobilized pH gradients
Authors: Kilias Harald , Gelfi Cecilia , Righetti Pier Giorgio
Published: May 2005

A base for a modern species' concept of chlorococcal algae can be obtained, not by morphological analysis, but by biochemical characters, e. g. isoenzyme banding patterns. From isolated lichen algae of the genus Trebouxia de Puymaly a set of five such enzymes has been studied by isoelectric focusing in immobilized pH gradients (IPG): phosphoglucomutase, phosphoglucose isomerase, malate dehydrogenase, mannitol dehydrogenase and leucine aminopeptidase. The first four are resolved into isoforms in a pH 4–7 IPG interval, while the last one is analyzed in an IPG pH 3.5–5 span. The patterns are specific for distinct populations, inter‐ and intraspecifically varying in dependence from their geographical distribution or the lichen species from which they have been isolated. Their limited heterogeneity (one to four isoforms) suggests that they are the products of specific genes rather than artefacts of the extraction procedure or the IPG analysis. Sharp isozyme patterns can only be obtained in a mixed‐bed, carrier‐ampholyte (CA)‐IPG gel and by anodic application, suggesting that the recently proposed mechanism of hydrophobic protein‐IPG matrix interaction (Electrophoresis, 1987, 8, 62–70) is fully operative here. As an additional mechanism, it is proposed that, in some cases, CA might simply act, when added to an IPG gel, by buffering, in the transient state, the sample zone before the protein migrates from the liquid phase into the IPG matrix.
Radically simplifying the Henry function
Authors: Michov Budin M.
Published: May 2005

We recently established that every ion has two radii – a geometrical one and an electrokinetic one. Using this principle, and in accordance with Henry's method of determining his function, f(κa), we have discovered that the Henry function can be radically simplified. The graphs of the proposed equation are plotted at different argument values. They are identical with the graphs plotted according to the Henry function.
Purification of cellulases from Streptomyces strain A20 by electroendosmotic preparative electrophoresis
Authors: Curioni Andrea , Peruffo Angelo Dal Belin , Nuti Marco P.
Published: May 2005

Five cellulase components were identified and purified in one step from Streptomyces strain A20 using electroendosmotic preparative electrophoresis. By this procedure up to 18 mg of protein mixture could be loaded on the column, with an estimated recovery of 60–70% of total activity; activity and protein recovery could be estimated 32% and 47%, respectively, if only activity peaks were considered. In comparison to other purification methods, this technique results in high protein recovery and resolution of applied samples.
Rapid imaging, using a cooled charge‐coupled‐device, of fluorescent two‐dimensional polyacrylamide gels produced by labelling proteins in the first‐dimensional isoelectric focusing gel with the fluorophore 2‐methoxy‐2,4‐diphenyl‐3(2H)furanone
Authors: Jackson Peter , Urwin Valerie E. , MacKay Craig D.
Published: May 2005

A new method for visualising proteins in two‐dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2‐methoxy‐2,4‐diphenyl‐3(2H)furanone (MDPF) while present in the first‐dimensional gel after isoelectric focusing and subsequently electrophoresed into the second‐dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge‐coupled‐device was used to view the two‐dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two‐dimensional gels at the end of electrophoresis with no further processing.
Characterization of human skin fibroblast extracellular proteins by two‐dimensional polyacrylamide gel electrophoresis
Authors: Graham Colin A. , McLean W. H. Irwin , Hughes Anne E. , Nevin Norman C.
Published: May 2005

Human skin fibroblasts secrete over 50 proteins into the culture medium. In this paper these are characterised using two‐dimensional polyacrylamide gel electrophoresis and peptide mapping of proteins metabolically labelled in the presence and absence of tunicamycin. Thirty of these proteins have been shown to be N‐glycosides, 4 are O‐glycosides and 10 are not glycosylated. Of the major proteins, groups 1–4 have previously been shown to be fibroblast specific. Peptide mapping and tunicamycin treatment has identified that groups 1 and 2, and 3 and 4 are closely related and that groups 1 and 3 arise by N‐glycosylation of 2 and 4, respectively. The unglycosylated precursor forms of several other proteins have also been identified. This approach to the analysis of protein secretion provides an abundance of information on many proteins simultaneously and can be used to assess the changes in protein secretion associated with development, and to identify extracellular growth factors and other regulatory proteins.
Characterization of genetic variants of human serum transferrin by isoelectric focusing: Comparison between conventional and immobilized pH gradients, and application to a protocol for paternity testing
Authors: Pascali Vincenzo L. , Dobosz Marina , Destro‐Bisol Giovanni , D'Aloja Ernesto
Published: May 2005

The polymorphism of transferrin (Tf) is currently being studied by isoelectric focusing in carrier ampholyte‐generated pH gradients, carrier ampholyte‐separator pH gradients or in immobilized pH gradients. Details for obtaining reproducible results with each of the three procedures are outlined. The effectiveness of pretreatment of serum samples with ferrous/ferric salts is discussed, and incubation times optimized after spectrophotometric measurement of the monoferric Tf conversion. Most of the presently available commercial batches of carrier ampholytes do not reliably discriminate the six common TfC subtypes. Resolution of C1, C3 and C2 was achieved by adding 20 to 90 mM HEPES slab gels prepared with various carrier ampholytes. Isoelectric focusing in carrier ampholyte‐separator pH gradients cannot be recommended as a standard typing procedure because the results strongly depend on the batch of carrier ampholytes. Tf subtype resolution was only achieved by using isoelectric focusing in immobilized pH gradients with pH slopes reliably reproducible from one experiment to another. Two major shortcomings of immobilized pH gradients are a marked tendency to protein precipitation at the application site and an interaction of proteins with the charged matrix. A protocol for Tf subtyping in immobilized pH gradients is described, based on prior desialylation of samples instead of pretreatment with iron. Sample entry into the matrix was optimized by addition of 5 mM Tris to the gels, and initially running them at low voltage. Recommendations are provided for the application of Tf typing for paternity testing.
Factor B(BF) subtyping by isoelectric focusing: Methods, nomenclatures, genetics and forensic application
Authors: Geserick Gunther , Patzelt Dieter
Published: May 2005

Usually factor B (BF) typing is performed by means of the traditional agarose gel electrophoresis. Using isoelectric focusing, the system can be extended by two common subtypes of BF F. The existence of BF F subtypes has in the meantime been confirmed by various authors and in different populations. Their inheritance has been proven by family‐and mother/child analyses and molecular‐genetic studies (correlation with restriction fragment length polymorphism). Different typing methods as well as different nomenclatures seem to indicate that the subtypes FA and FB (according to Gesericket al.) are identical with the Fb and Fa subtypes (according to Teng and Tan). At present, some confusion still exists for the less frequent variants and subtypes which possibly could be identified by direct comparison of the patterns. The BF system is a valuable marker in patennity testing. Its chance for exclusion of paternity in Caucasian populations has been calculated to be about 14 % for agarose gel electrophoresis and increases to about 16 % for BF F subtyping by isoelectric focusing. Preliminary results indicate that BF may also be used for typing of bloodstains (up to 2 weeks old).
A new sample applicator for isoelectric focusing in horizontal polyacrylamide gels
Authors: Altland Klaus , Becher Pia
Published: May 2005

A new sample applicator for horizontal flat gels is described. The applicator is practically safe against contamination from adjacent samples and can be used for all types of electrophoretic separations including a concentration step for either the sample (i. e.disc electrophoresis) or the separated zones (i. e.isoelectric focusing). The applicator is a piece of flat glass with 26 or 51 parallel 2 mm wide grooves, drilled at distances of 9 or 4.5 mm. Samples, maximally 25 or 50. are applied to the areas between the grooves. By inverting the applicator, the samples are brought into close vicinity to the gel surface and the pendant droplets expand by capillary attraction into the slits between the glass and gel with resultant even distribution across the lanes of 2.5 or 7 mm width. The applicator can be used for separations with and without protection of the electrophoretic setup by paraffin oil and allows for fast multiple handling of samples by means of appropriate syringes and microtiter plates.
Electroblotting of Immobiline DryPlates applied to identification of human plasma apolipoprotein A‐I
Authors: Holmquist Leif
Published: May 2005

A method for efficient electroblotting of Immobiline DryPlates, allowing subsequent immunological identification of separated proteins has been developed. A thin layer of 1 % agarose containing sodium dodecyl sulfate is moulded on the 0.5 mm thick polyacrylamide gel surface after completed electrophoresis. After separation of the agarose‐polyacrylamide gel sandwich from the plastic film the rigid gel sandwich could be easily transferred to a nitrocellulose membrane and electroblotting could be performed without adherence of the sticky polyacrylamide gel layer to the membrane. Using this technique human plasma high density apolipoprotein A‐Iisoforms, over a wide concentration range, could be identified in a heterogeneous mixture, conserving the isoform pattern and band sharpness produced in the immobilized pH gradient experiments.
Immobilized pH gradients in ultrathin‐layer (100 μm) gels: Avoiding capillarity by a ‘squeezing‐sealing mold’ technique
Authors: Pascali Vincenzo L. , Dobosz Marina , D'Aloja Ernesto
Published: May 2005

Several procedures for casting 100 μm ultrathin immobilized pH gradients are described. When acrylate/glass molding cassettes (Pascali et al., Electrophoresis 1987, 8, 371–373) are used two main problems are encountered: (i) a tendency of polymerization solutions, at the beginning of delivery, to spread across the glass surfaces with troublesome effects on the gradient stratification, and (ii) the raising of steep menisci at both extremities of the pH intervals, originating from capillarity phenomena and resulting in nonuniform gradients with bowed electrophoretic patterns. The first shortcoming was acceptably solved by increasing the density of sucrose gradients, and pouring them into prewarmed molding cassettes. The detrimental effect of menisci could be overcome by using a ‘squeezing‐sealing mold’ technique. A molding cassette was endowed with a continuous, squared spacer frame, the upper side being open by inserting a wedged clip. A slight excess of polymerization solution was first dispensed into the cassette and squeezed away on removal of the clip. By completely excluding air from the molding cassette, uniform and well reproducible ultrathin gels could be cast. A major advantage of ultrathin immobilized pH gradient gels is the drastically shorter focusing time.
Two‐dimensional electrophoresis. Two‐dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes
Authors: Asakawa Jun‐ichi
Published: May 2005

Two‐dimensional electrophoresis (2‐DE) with immobilized pH gradient (IPG) gels in capillary tubes was used in the first‐dimensional isoelectric focusing (IEF) for the separation of human platelet polypeptides. Two types of IPG tube gels, pH ranges 4–8 and 7–10, containing 8 M urea, 1% Nonidet P‐40 and 0.1% pH 3.5–10 Ampholine carrier ampholytes (CA) were prepared by a simple method not requiring special equipment. The addition of CA to both gel and sample solutions was essential in the tube gel IPG system. Proteins were visualized by a modification of Wray's silver‐staining technique. The degree of resolution and the number of spots observed on an IPG 2‐DE gel with pH 4–8 were comparable with those obtained with O'Farrell's high‐resolution 2‐DE. Approximately 200 basic polypeptides, which are difficult to separate by conventional CA‐based IEF 2‐DE or the non‐equilibrium pH gradient system, were well resolved by 2‐DE with a pH 7–10 IPG tube gel in the first‐dimension. The gel patterns with either pH gradient 4–8 or 7–10 were highly reproducible among gels prepared and run simultaneously. These results demonstrated the potential and usefulness of the 2‐DE system with IPG gels in capillary tubes.
Microheterogeneity of human plasma lecithin:cholesterol acyltransferase examined by isoelectric focusing in immobilized pH gradients
Authors: Holmquist Leif , Bjellqvist Bengt
Published: May 2005

The microheterogeneity of highly purified human plasma lecithin:cholesterol acyltransferase (LCAT) has been examined by electrophoresis in immobilized pH gradients in Immobiline‐polyacrylamide gels of the pH ranges 4–7 and 4.2–4.9. Seven isoforms were obtained with LCAT isolated from pools of normal plasma. Using this technique the apparent pI values at 15 °C for the isoforms in the pH 4.2–4.9 gradient were 4.37, 4.42, 4.48, 4.53, 4.60, 4.67 and 4.74. (SD = ± 0.03 for all). The most intensely stained band in the isoform pattern corresponded to the isoform with a pI value of 4.48.
Immobilized pH gradient isoelectric focusing of cerebrospinal fluid proteins
Authors: Pirttilä Tuula , Frey Harry , Mattila Kari , Sidén Åke
Published: May 2005

Isoelectric focusing in immobilized pH gradients, supplemented with 0.5 % w/v carrier ampholytes was applied for studies of native proteins, especially immunoglobulin G, in cerebrospinal fluid and serum. All 72 paired samples were run on pH 4–10 gels; 25 of them were also examined in pH 7–10 gels. Silver staining and nitrocellulose blotting with amplified immunoperoxidase detection of immunoglobulin G were used for protein visualization. Intrathecally produced immunoglobulin G was resolved into sharply focused, straight and easily identifiable fractions. The pH gradients were stable and the inter‐gel reproducibilities of individual immunoglobulin G patterns were good.
Subtyping of group specific component (GC) in human semen, blood and vaginal fluid by isoelectric focusing in immobilized ph gradients
Authors: Pötsch‐Schneider Lucia , Klein Hartmut
Published: May 2005

The group specific component (GC) is stable and well suited for forensic casework. Isoelectric focusing of common GC variants from semen, seminal fluid, vaginal fluid and semen stains, on Immobiline DryPlates, pH 4.5–5.4, is of practical value in criminal investigations of sexual deliquencies. GC is present in normospermia and azoospermia seminal fluids and found in about 20 % of the vaginal secretions. The GC patterns observed were similar and in accordance with the bands of the individual GC type in plasma/serum.