bluuebunny/crossref_metadata_2025 huggingface dataset:
Natural pH gradients formed by two and three aminoacids on polyacrylamide gel: Changes of pH, segmental voltages and aminoacid distributions with time
Authors: Jackiw B. Andrew , Chrambach Andreas
Published: May 2005

A natural pH gradient can be formed on polyacrylamide gel using either two (glutamic acid and lysine only) or three (glutamic acid, histidine and lysine) aminoacids as the sole carrier ampholyte species. In the two component system, glutamic acid and lysine focus as single peaks adjacent (anodically and cathodically, respectively) to the steep, linear center of the step‐function pH gradient. Histidine, in the three component system, focuses at the center of the pH gradient and separates the other two aminoacids. Each aminoacid electrofocuses at its pI. Hemoglobin electrofocuses as a sharp band in both systems and, in the two component system, remains focused at its pI for 200 h. A major conductance gap forms at the gel center in both systems with progressing time of electrofocusing; a minor and transient conductance gap develops in the glass tube just above the gel.
The analysis of the proteins of heterogeneous ribonucleoprotein particles on two‐dimensional polyacrylamide gels
Authors: Wilks Andrew F. , Knowler John T.
Published: May 2005

Methods of two‐dimensional protein fractionation have been analysed for their suitability for the analysis of heterogeneous ribonucleoprotein (hnRNP) particles. Nonequilibrium pH gradient electrophoresis gives the best overall fractionation and reveals that the dominant “core” proteins possess charge heterogeneity. For the analysis of the minor components of the particles, it may be preferable to overload isoelectric focusing gels and allow the basic core proteins, which comprise 70% of the total protein, to migrate off the gel. This does, however, result in the loss of some basic minor polypeptides.
The “betagram” for rapid localization of 3H‐labeled proteins in SDS polyacrylamide gels
Authors: Kruppa Joachim , Bauche Andreas
Published: May 2005

The distribution pattern of 3H‐labeled proteins in slab gels has been recorded with two instruments: a thin‐layer scanner and a Betacamera, both of which have been used with success previously in monitoring paper and thin‐layer chromatograms. Individual radioactive protein bands may be localized with both instruments although they differ markedly in sensitivity. The scanner is relatively insensitive and thus time‐consuming, since each lane of the gel has to be recorded separately. In contrast, with the Betacamera the whole area of the slab gel could be monitored simultaneously. The extraordinarily high sensitivity of the Betacamera in detecting 3H‐compounds via the spark chamber proved to be of great advantage since we intended to isolate preparatively specific proteins from the gel. The “betagram” technique has a very low detection limit and is thus less time‐consuming than fluorography. However, the resolution of the “betagram” is limited due to the number of wires in the spark chamber, and does not reach the quality attained with fluorography.
Amplified immunoperoxidase staining of isoelectrically focused human tumor markers
Authors: Saravis Calvin A. , Cantarow Walter , Marasco Patrick V. , Burke Bohdana , Zamcheck Norman
Published: May 2005

Avidin‐biotin amplified immunoperoxidase staining of the human tumor marker, carcinoembryonic antigen (CEA) localized in malignant tissue by isoelectric focusing is described. CEA either in electrofocused aqueous tissue extracts or extracted and separated by direct tissue isoelectric focusing in agarose (IsoGel), was reacted with rabbit and anti‐CEA antiserum and with enzyme conjugated swine anti‐rabbit antiserum, or with avidin‐biotin conjugated reagents, then with enzyme substrate. Avidin‐biotin amplified immunoperoxidase staining of CEA gave more intense staining. Quantitation and characterization of tumor markers by direct tissue electrophoresis in isoelectric focusing media and enzyme amplification of anti‐tumor marker antisera, make possible the study of their biological meaning and clinical usefulness.
Two‐dimensional micro‐electrophoresis of proteins from plant‐parasitic nematodes: Increased sensitivity of protein detection by silver staining or in vivo labeling
Authors: Poehlingss Hans‐Michael , Wyss Urs , Neuhoff Volker
Published: May 2005

Minimal amounts of proteins from plant‐parasitic nematodes are sufficient for separation in two‐dimensional micro‐electrophoresis with high resolution. It can be shown that the host – parasite interaction leads to a drastic change of the protein composition in the parasite. Silver staining or in vivo labeling of the nematode proteins results in an increased sensitivity of protein detection.
Demonstation of human prealbumin by double one‐dimensional slab gel electrophoresis
Authors: Altland Klaud , Rauh Silke , Hackler Rolf
Published: May 2005

A double one‐dimensional (D 1‐D) electrophoretic method for slab gels with polyacrylamide gel electrophoresis (PAGE) followed by polyacrylamide gel isoelectric focusing (PAGIF) is described for the demonstration of prealbumin (PA) from human sera. Almost pure PA is obtained when serum is submitted to Page i an anionic discontinuous (glycine‐chloride) buffer system at alkaline pH. After page the Bromophenol Blue‐stained tracking dye boundary in the gel containing PA is cut out and the gel strips are overlayed on an isoelectric focusing gel. A final PAGIF pattern of PA is obtained which is hidden by other serum proteins when one‐dimensional PAGIF is performed. PAGIF of PA in a gel containing a gradient from 0 to 8 M urea perpendicular to the pH axis reveals the conversion of at least 7 diffuse bands with isoelectric point (pI) values between 4.2 and 4.7 into three sharp zones with pI values of 5.45 for one minor zone and 5.7 for the two other zones lying very close together. Among 1900 sera from adult pregnant women, 2 were found containing a genetically determined PA variant with a pI of 5.9 for a minor and a pI of 6.35 for a more intense zone in addition to the usual pattern. From the variant PAGE and PAGIF patterns and their distribution in the families of the carriers, it is concluded that PA is a tetramer under control of an autosomal structural gene which remains stable during PAGE abut dissociates into the monomers during PAGIF in the presence or presence of urea it is followed that PA under physiological conditions possibly forms complexes with more components than expected from its known binding capacity for thyroxine and retinal binding protein (RBP). This highly resolving K 1‐D electrophoretic technique allows the simultaneous analysis of up to 96 samples under identical experimental conditions. It is concluded that PA may be a good candidate for human mutation monitoring at the protein level.
The use of carbamylated charge standards for testing batches of ampholytes used in two‐ dimensional electrophoresis
Authors: Tollaksen Sandra L. , Edwards Jesse J. , Anderson Norman G.
Published: May 2005

A method of testing batches of ampholytes is presented. By using carbamylated charges standards to co‐electrophorese with the protein sample in the first‐dimension isoelectric focusing gel, one can monitor, after running and staining the second‐dimension sodium dodecyl sulfate (SDS) slabs gel, the continuity of the pH gradient. Charge standards can also be used to check the reproducibility of the pH gradient among batches of ampholytes and to modify the new batch with a small amount of a narrow range ampholyte to assure reproducibility of experiments. Ampholytes for comparison were obtained from three major manufacturers.
Toward an improved immunoglobulin analysis by gel electrophoresis and electrofocusing
Authors: Chrambach Andreas , Lan Birgit An Der , Mohrmann Helga , Felgenhauer Klaus
Published: May 2005

The analysis of immunoglobulin by gel electrophoretic methods is presently burdened with two problems: the incapacity of conventional buffer systems to spread the polyclonal immunoglobulin pattern across the entire separation path length in gel electrophoresis, and the appearance of distinct bands in the conventional electrofocusing patterns, instead of he continuum of zones — which would be expected form a polyclonal mixture of “charge isomers” and which is indeed obtained in gel electrophoresis. Polyclonal serum immunoglobulin differ from one another predominantly in net charge (i.e., they are “charge isomers”). The operative pH of gel electrophoresis was therefore optimized. The acidic side of he isoelectric operative pH of 5.32, a discontinuous (multiphase) buffer system was found which was capable of stacking all immunoglobulin in a lyophilized preparation.

In order to provide a magnified view immunoglobulin heterogeneity, the immunoglobulin mixture was fractionated into 26 groups of charge isomers using preparative isotachophoresis. When subjected to polyacrylamide gel electrophoresis in resolving gels operative at pH 4.85, 0°C, the isotachophoretic fractions spread between relative mobility values of 0.4 to 0.9 in the electrophoresis pattern, i.e., their sum total covered the entire separation path. Under these conditions, isotachophoretic fractions also revealed immunoglobulin oligomers. The number of oligomeric forms increased from increased from the leading (most basic) to the trailing (most acidic) immunoglobulin components within the stack.

In polyacrylamide gel electrofocusing of the isotachophoretic fractions of immunoglobulin, using pI‐renge 6–9 ampholine, addition of a 0.01 M mixture of amino acids to the carrier ampholytes, and 20h or more electrofocusing time at 20 V/cm of gel, 0°C, polyclonal immunoglobulin patterns appeared continuous. Electrofocusing patterns of those isotachophoretic fractions of immunoglobulin which contained oilgomeric forms (as revealed by gel electrophoresis ) exhibited one or two major bands against the continuous background of stainable components. Monoclonal immunoglobulin produced distinct major bands under the same comditions.

Thus, either polyacrylamide gel electrofocusing in the pI‐range 6–9 or polyacrylamide gel electrophoresis in a buffer system operative at pH 4.85 appear promising for the development of a clinically practical and artifact‐free electrophoresis analysis for specific oligoclonal immunoglobulin.
Agarose‐Sephadex: A new improved matrix for preparative flatbed isoelectric focusing
Authors: Manrique Albert , Lasky Marvin
Published: May 2005

A new, simple procedure is described for the preparation gel matrix for preparativde isolelectic focusing (IEF). 0.5–1% Agarose of IEF grade is incorporated with 2.5% Sephadex G‐200 Superfine (SF) and 3% Pharmalyte, yielding a uniform and rifid m atirx. No gel washing, swelling of degassing is necessary. Optimum gel consistency is quickly and easily obtained since no dehydration of the gel timum gel consistency is quickly and easily obtained since no dehydration of the gel slurry is required. Gel preparation time is reduced to 0.5–1 h and gel handling is simplified. Protein can be easily recovered from the matrix. The pH gradient profile and resolving properties of agarose‐Sephadex were compared with those of Spphdex G200 SF and IEF‐grade agarose. The profiles of agarose‐Sephadex and Sephadex matrixes are smoother and extend further in both directions than those for agarose. Focusing in agarose‐Sephadex and Sephadex also results in fewer edge effects. The ability to distinguish closely resolved bands in agarose‐Sephadex is superior due to its ability to be directly stained and its avoidance of eletroen‐domestic effects associated with focusing in sgarose. Changes in voltages enables us to follow the development, stability, and decay of the pH gradient. Agarose‐Sephadex and Sephadex pH gradients developed more smoothly and remained more stable than those for agarose. This new support system combines the good focusing properties of Sephades G‐200 SF with the simple handling, rapid and uniform gel formation of agarose, while overcoming many of the drawbacks of using either material alone.
Agarose gel electrophoresis of bacteriophages and related particles. II. Correction of electrophoretic mobilities for the electro‐osmosis of agarose
Authors: Serwer Philip , Hayes Shirley J.
Published: May 2005

Bacteriophage T4, bacteriophage T4 without its tail fibers, and bacteriophage P22 without its tail have been subjected to electrophoresis in agarose gels; electrophoretic mobility (μ) of these particles as a function of the concentration of agarose (A) has been determined using preparations of agarose with different amounts of electro‐osmosis. When μ was extrapolated to an A of 0 (μo), it was found that a term, μE, constant for each preparation of agarose must be added to μo to correct for electro‐osmotic flow. A relationship between μE and a manufacturer‐determined coefficient of electro‐osmosis has been empirically derived. Corrected μo's are in agreement with data obtained by solid support‐free electrophoresis.
Periodic acid‐Schiff staining of type II collagen and its cyanogen bromide peptides after polyacrylamide gel electrophoresis
Authors: Rajan Jess C. , Klein Leroy
Published: May 2005

Pepsin‐solubilized type II collagen and some of its CNBr‐derived peptides could be stained for carbohydrate with periodic acid‐Schiff (PAS) after polyacrylamide gel electrophoresis. PAS intensely stained an aldehyde‐containing peptide (Mr > 10 000) which was not detected with Coomassie Brilliant Blue R‐250 stain. Types I and III collagens and their CNBr peptides stained poorly, if at all, with PAS.
Isoelectric focusing of α1‐antitrypsin (PI) using restricted pH‐range carrier ampholytes in combination with a highly cross‐linked gel and a separator
Authors: Klasen Eduard C. , Rigutti Adriana
Published: May 2005

A simple technique for polyacrylamide gel isoelectric focusing of α1‐antitrypsin (PI) using thin, highly cross‐linked gels, restricted pH‐range carrier ampholytes and a separator was developed. This method enables easy typing of all known PI phenotypes including the four PI*M subtypes.
Isoelectric focusing studies of antibody clonotype patterns in outbred rabbits during prolonged immunization
Authors: Binion Steven B. , Rodkey L. Scott
Published: May 2005

Isoelectric focusing was used to study the antibody clonotype patterns of outbred New Zealand White rabbits during pronlonged immunization with the gram positive bacterium Micrococcus lysodeikticus. Antisera were examined by analytical isoelectric focusing in 1% agarose gels using inexpensive carrier ampholytes which were synthesized in the laboratory. Clonotype patterns of antimicrococcal antibodies were visualized by radioautography using [125I]‐labeled micrococcal carbohydrate antigen. Examination of the antibody clonotype patterns in antisera from a number of outbred rabbits revealed that variations in clonotype expression often occurred within an individual during successive rounds of immunization. These studies demonstrate the utility of analytical isoelectric focusing for monitoring antibody clonotype changes within an individual during immunization.
2‐D Electrophoresis gel data base analysis: Aspects of data structures and search strategies in GELLAB
Authors: Lemkin Peter F. , Lipkin Lewis E.
Published: May 2005

Search strategies for finding spot differences among multiple two‐dimensional (2‐D) polyacrylamide electrophoresis gels are discussed in the context of the GELLAB spot data base management system. A 2‐D gel experiment should have a well‐defined biological experimental and preparation protocol reflecting the hypotheses of the problem. So too should the analysis of its corresponding 2‐D gel computer spot data base have a protocol. This protocol is heavily influenced by the nature of the biological experiment as well as 2‐D gel preparation considerations including the realities of artifactual and systematic noise. It is further influenced by constraints due to computational considerations. The search strategy is that part of the analysis protocol in which an experimenter iteratively defines tests to find significant spot differences. One goal of designing a well thought out search protocol is to reduce the number of search iterations required. Aspects of some requirements and constraints for useful search strategies are discussed.
Measurement of amino acid composition of proteins by computerized microdensitometry of two‐dimensional electrophoresis gels
Authors: Latter Gerald I. , Metz Edward , Burbeck Stephen , Leavitt John
Published: May 2005

A method which utilizes computerized microdensitometry to measure amino acid compositions of polypeptides resolved in two‐dimensional gels is described. Twenty cultures of two human cell lines were radiolabeled with each of the 20 different 14C or 35S – amino acids. Two‐dimensional polyacrylamide gel electrophoresis was performed on total protein samples of the cultures in replicates of three. For each amino acid label the relative amount of radioactivity in an “unknown” polypetide species was computed using the measured radioactivity simultaneously incorporated into the reference protein, β‐, γ‐actin, whose precise amino acid composition is known. We tested the accuracy of this method by computing the amino acid composition of two species of mutant β‐actin, which along with normal β‐, γ‐actin as the reference provided us with an ideal set of conditions. We found that amino acid composition can be predicted with a standard error of 6% over all twenty amino acids. Techniques are described to further reduce the measurement error so that compositions of proteins can be measured under less ideal conditions.
Direct digital imaging of radio‐labeled two‐dimensional gel beta emissions using micro‐channel plate image enhancement
Authors: Burbeck Stephen
Published: May 2005

The use of autoradiography to record the distribution of radio‐labels such as tritium or 35S in two‐dimensional (2‐D) gels has well known deficiencies: the film requires long exposures, has a nonlinear response curve, and has a restricted dynamic range. Autofluorography can improve upon each of these problems but only at the cost of resolution and quantitative accuracy. This paper demonstrates that a micro‐channel plate electron multiplier and a resistive anode position detector can detect and localize individual beta emissions from 35S or tritium with at least 200 μm resolution. The locations of these beta emissions can be collected, converted to a 2‐D image, and displayed on a graphics terminal or integrated over spot areas for quantitative estimates of spot intensity. The collection process is rapid, linear, and has a dynamic range far greater than that of film. With the 35S‐test gel (which required 40 h for an autoradiogram), strong spots can be integrated with better than ten percent accuracy with less than five minutes of data collection. Weak spots (those with less than 4 cpm) may require 80 min or more. Tritium gels require collection times an order of magnitude longer for equivalent quantitative accuracy.
Two‐dimensional polyacrylamide gel electrophoresis in micro‐gels: Gradient gel electrophoresis followed by dodecyl sulfate gel electrophoresis
Authors: Beltle Wolfgang , Zimmermann Sabine , Bopp Martin , Murach Karl‐Friedrich
Published: May 2005

A miniature system for two‐dimensional polyacrylamide gel electrophoresis is presented. Discontinuous sodium dodecyl sulfate gel electrophoresis in micro‐slab gels following high resolution micro‐electrophoresis in cylindrical continuous polyacrylamide gradient gels separates macromolecules after an initial size fractionation into their subunits. The construction of a glass cuvette for micro‐slab gels and of a suitable electrophoresis tank is described in detail. Furthermore, procedures for silver staining and fluorography initially developed for gels of normal size were adapted to micro‐slab gels. Crude tissue extracts of Funaria hygrometrica L. Sibth. were analyzed without any further purfication. Twenty samples can be processed in about eight hours.
Isoelectric focusing in immobilized pH gradients for the determination of the genetic Pi (α1‐antitrypsin) variants
Authors: Görg Angelika , Postel Wilhelm , Weser Johann , Weidinger Sebastian , Patutschnick Walburgis , Cleve Hartwig
Published: May 2005

The genetic variants of the human protease inhibitor (Pi) system have been analyzed by isoelectricfocusing in immobilized pH gradients. With the use of a narrow pH range of pH 4.50 to 4.70 the six common PiM subtypes can be readily and reliably classified. The less common inherited Pi variants can be demonstrated most clearly in a gradient of pH 4.4 to 4.8. For the classification of PiZ a more basic pH gradient of 4.55 to 5.05 has been employed. Isoelectric focusing with immobilized pH gradients is also a useful tool for the study of the genetic variation in the Pi system. A number of problems to be adressed in future studies are discussed such as the delineation of further PiM subtypes, the detection of additional inherited heterogeneity, and the application of isoelectric focusing with immoblilized pH gradients in routine typing laboratories.
Selected buffer systems for moving boundary electrophoresis on gels at various pH values, presented in a simplified manner
Authors: Chrambach Andreas , Jovin Thomas M.
Published: May 2005

Moving boundary electrophoresis on gels is useful for 4 purposes: (1) As an automatic concentration method to produce concentrated starting zones of proteins* for analytical and preparative resolving gels; (2) to provide a sharply defined reference zone in the resolving gel for Rf measurement; (3) for the preparative electrophoretic extraction of proteins from gel slices; (4) for high load preparative “charge fractionations” of relatively simple mixtures of proteins. It requires buffer systems at various pH values which are capable of setting up moving boundaries with numerically known leading and trailing ion mobilities. Moreover, since protein migration is slow compared with that of buffers, these moving boundaries have to have low displacement rates if they are to concentrate and align the proteins in order of mobility. Ten years ago, the authors made available in form first of magnetic tapes, then of microfiche, a collection of buffer systems for that purpose. But, in spite of the large number, over 4000, of these buffer systems, biochemists have not taken advantage of them widely. This appears in part due to the terminological complexity of the presentation needed to give an exhaustive physical‐chemical description of the systems. In part, it was due to the confusion about which of the various forms and techniques of moving boundary electrophoresis, known under a variety of names, these buffer systems applied to. Also, among the many buffer systems, only relatively few exhibited sufficiently low moving boundary displacement rates to allow slowly migrating proteins to migrate within the moving boundary. Finally, it proved difficult for potential users to interpret the magnetic tapes and microfiche; also tapes were excessively expensive. To remedy all these problems, and to allow biochemists to benefit more widely, both analytically and preparatively, from moving boundary electrophoresis at any pH, we have selected a small number of buffer systems capable of concentrating proteins within moving boundaries set up at various pH values, with low displacement rates, and with either anodic or cathodic displacement. These are presented in simplified format and terminology.
Direct isoelectric point determination of antigens by a new quantitative immunoelectrophoretic method
Authors: Guinet Ronald M. F.
Published: May 2005

Crossed immunoelectrofocusing combines the high resolution of isoelectric focusing in the first dimension with the detection selectivity of quantitative immunoelectrophoresis through an antibody‐containing gel in the second dimension. A new quantitative immunoelectrophoretic method is described which permits in a single process simultaneous determination of isoelectric points and identification of individual proteins in a complex mixture. This method combines crossed‐line immunoelectrofocusing with crossed‐line immunoelectrophoresis on the same plate. Optimal conditions presented have been determined with our Candida tropicalis antigen‐antibody system.
Demonstration of activity for alkaline muscle ATPase in polyacrylamide gels using metal conversion methods
Authors: Kirkeby Svend , Moe Dennis
Published: May 2005

ATPase activity from skeletal muscle was tested at pH 9.2 in polyacrylamide gels using cobalt or lead conversion techniques. With cobalt it seems that the ion is precipitated both to acrylamide and to certain muscle components resulting in a reproducible but non‐enzymatic banding pattern. With the lead technique a specific strongly coloured band is present near the cathode. The band is suppressed by p‐hydroxymercuribenzoate but not by levamisole.
Two‐dimensional gel electrophoretic analysis of plasma proteins from a patient bitten by a rattlesnake
Authors: Bagley Elisabeth A. , Lonberg‐Holm Karl , Pandya Bharat V. , Budzynski Andrzej Z.
Published: May 2005

We have studied changes in plasma proteins in a patient bitten by a western diamondback rattlesnake (Crotalus atrox). Samples obtained at intervals during recovery were analyzed by “low resolution” two dimensional (2‐D) gel electrophoresis, a method which resolves at least 50 plasma components. The results show three days of complete afibrinogenemia, followed by gradual return of fibrinogen to a normal level. Both α‐ and β‐ lipoproteins also appeared altered or decreased in concentration during this period. An anomalous change in electrophoretic mobility of a portion of complement component 3 (C3) was detected during the first 15 days, but not at day 38. This modified protein was not C3b or C3c. A short incubation of human plasma or purified C3 with venom in vitro, did not mimic in vivo effects on either fibrinogen or C3, but plasma lipoproteins were modified directly in vitro. It is possible that the in vivo effects of venom on C3 and fibrinogen, as revealed by the 2‐D gel electrophoretic method, are indirect and require release or activation of cellular proteases.
Identification of mouse serum proteins increased by the administration of antitumor polysaccharide lentinan, by micro two‐dimensional electrophoresis
Authors: Manabe Takashi , Takahashi Yuko , Okuyama Tsuneo , Maeda Yukiko Y. , Chihara Goro
Published: May 2005

The changes in mouse serum protein distribution after administration of lentinan, an antitumor polysaccharide, were examined using micro two‐dimensional electrophoresis in the absence of denaturing agents. Three protein spots were markedly increased in level and two of them were identified to be haptoglobin and haptoglobin partially bound with hemoglobin. Activity staining of haptoglobin after complex formation with hemoglobin and of ceruloplasmin were demonstrated.
The use of polyacrylamide gradient gel electrophoresis for the quality control of lectins
Authors: Caron Michel , Faure Alain
Published: May 2005

Polyacrylamide gradient gel electrophoresis is a sensitive and high resolution method for the characterization of commercial peanut lectin preparations. It is suitable for the detection of aggregates in lyophilized samples.
Measurement of isoelectric point of S‐adenosyl‐L‐methionine and its metabolic products by an isoelectric focusing technique
Authors: Farooqui Jamal , Kim Sangduk , Paik Woon K.
Published: May 2005

The isoelectric point (pI) of S‐adenosyl‐L‐methionine and some of its metabolic products has been determined by an isoelectric focusing technique. The experimental pI values of S‐adenosyl‐L‐methionine and S‐methylmethionine are 7.24 and 6.85, respectively, and the pI values of S‐adenosyl‐L‐homocysteine and homocysteine are 6.05 and 5.65, respectively. This indicates that the contribution by adenosine, adenine and ribose to the total electric charge of S‐adenosyl‐L‐methionine or S‐adenosyl‐L‐homocysteine is approximately 0.40 pI unit. The differences in the pI values between two pairs of compounds (S‐adenosyl‐L‐methionine vs. S‐adenosyl‐L‐homocysteine and S‐methylmethionine vs. methionine) are very close (1.19 and 1.14, respectively). This difference is the amount of pI value contributed by the sulfonium group which has been formed by methyl addition. Surprisingly, the presence of a permanent positive charge at the sulfonium center in S‐adenosyl‐L‐methionine and S‐methylmethionine does not result in a higher pI values for these compounds. This might be due to the partial neutralization of the positive charge by the formation of carbanion. The mechanism of charge neutralization in these sulfonium compounds is discussed.
Studies of silver staining of human parotid saliva protein polyacrylamide gel electrophoretograms
Authors: Hallinan Frank M.
Published: May 2005

The silver staining procedure of Oakley et al. [3] has been applied to parotid saliva proteins and certain protein standards fractionated by isoelectric focusing and electrophoresis in denaturing and non‐denaturing polyacrylamide gels. This staining method was up to 100‐fold more sensitive than Coomassie Blue staining for sodium dodecyl sulphate‐containing gels and for focusing gels, after prefixation. In non‐denaturing gels, however, the relative sensitivity (i.e. silver vs. Coomassie) of different bands varies widely but this can be overcome by silver staining of gels after Coomassie Blue staining.
Agarose gel electrophoresis of bacteriophages and related particles. IV. An improved procedure for determining the size of spherical particles
Authors: Serwer Philip , Allen Jerry L.
Published: May 2005

Attempts have been made to improve the accuracy with which sieving during electrophoresis in gels can be used to determine the size of viruses and related particles. Using as samples spherical bacteriophages and related particles of known radius (13.3–41.9 nm), it was found that electrophoretic mobility (μ) was a linear function of agarose concentration (A) in a hydroxyethylated agarose (SeaPlaque, Marine Colloids) for 0.4 ⩽ A ⩽ 1.6. The value of A extrapolated to a μ of 0 (A0) was a decreasing function of particle outer radius and was independent of the solid support‐free μ. Procedures for determining outer particle radius from values of A0 have been developed. When used with internal standards, these procedures measure outer particle radius with an accuracy of ±4 %.
Subtyping of human red cell phosphoglucomutase (PGM1) using conventional agarose gel electrophoresis
Authors: Bäur Walter , Dissing Jørgen
Published: May 2005

A simple, rapid electrophoretic method for the subtyping of phosphoglucomutase (PGM1) isozymes is presented. Electrophoresis was performed in an agarose gel containing an acetic acid/L‐histidine/citric acid buffer system at pH 5.30 for 2.5 h. The separation of the isozymes was at least as good as that obtained by isoelectric focusing, but in contrast with that method, the electrophoretic procedure did not require expensive equipment or carrier ampholytes.
Two‐dimensional electrophoresis of serum proteins in myocardial infarction patients and subjects at risk of developing coronary heart disease
Authors: Gomo Zvenyika A. R. , Clark Penelope M. S. , Kricka Larry J. , Woods Kent , Buckley Brendan , Whitehead Thomas P.
Published: May 2005

Iso‐Dalt analysis of serum from myocardial infarction patients has revealed the presence of a group of low (<23 000) molecular weight basic peptides, as yet unidentified, which were not found in the serum of healthy controls. The presence and staining intensity of these proteins correlated with the qualitatively assessed serum creatine kinase MB isoenzyme activity. The proteins were not detected in sera from patients at risk of developing coronary heart disease (smokers, hypertensive patients, subjects with a family history of developing coronary heart disease, oral contraceptive users, hyperlipidaemics, and diabetics). This excludes their possible use as a predictive test for the development of coronary heart disease. It seems probable that they originate in the cells in or near the infarct.
The titration curve of native C reactive protein
Authors: Laurent Philippe , Potempa Lawrence A. , Gewurz Henry , Fiedel Barry A. , Allen Robert C.
Published: May 2005

We have studied the electrophoretic mobility of purified human C reactive protein (CRP) in a gradient from pH 3.0 to 9.0 produced by the titration curve method. The results suggest that the major species has a pI at pH 6.3 ± 0.2. From pH 7.2 to 8.5, CRP was at its isoelectric point and did not migrate from the application trough. From pH 4.0 to 5.0, CRP produced a hazy, diffuse pattern most probably due to instability in the region close to the isoelectric point of the acid‐denatured form of the molecule. Below pH 4 CRP resolubilized to assume heterogenic anionic forms.
Separation of legumin and vicilin, the storage proteins from germinating Vicia faba seeds, by electrophoresis on cellulose acetate membranes
Authors: Leslie Philip , Bonner Richard , Vandevalle Ian , Olsson Richard
Published: May 2005

Conventional electrophoretic methods of assessing the purity of a protein are both expensive and laborious. The method of zonal isoelectric precipitation electrophoresis involves precipitation of the protein under investigation at its isoelectric point after application onto cellulose acetate membranes. Contaminating proteins will be mobile and are easily visualized by conventional staining methods. Application of zonal isoelectric precipitation electrophoresis to the storage proteins of Vicia faba seeds (legumin and vicilin) has proved valuable in this laboratory, and, by adjustment of the buffering system, can be made to work for other protein mixtures.
The effects of freezing and thawing on lactate dehydrogenase and esterase isozyme patterns of the goldfish Carassius auratus, demonstrated by isoelectric focusing
Authors: Burch Simon J. , Lawson Ronald , Davies David H.
Published: May 2005

Current literature suggests that the reliability of results obtained from electrophoretic studies using material which has been frozen is in doubt. Banding patterns of lactate dehydrogenase and non‐specific esterase produced by isoelectric focusing (IEF) of fresh tissues from the goldfish, Carassius auratus, are presented. Changes which occur in these patterns due to repeated freezing and thawing involve a reduction in both the intensity and the number of bands. Differences in the IEF profiles are extensive and consistent. Changes also occur in material which has been frozen for a ten‐week period and thawed once. Storage in liquid nitrogen, ‐180°C, as distinct from a conventional freezer at ‐20°C, produes no significant improvement. Interpretation of results from material that has been frozen must therefore be undertaken with extreme care, especially when sensitive separation techniques like IEF are used.
A rapid detection system for the quantitative evaluation of radioactively labeled proteins in dehydrated slab gels
Authors: Kruppa Joachim
Published: May 2005

3H‐ and 35S‐Labeled proteins of vesicular stomatitis virus were separated by polyacrylamide gel electrophoresis, localized and directly quantified by the Linear Analyzer after dehydration of the gel. The counting efficiency of the Linear Analyzer was approximately 0.1% for tritium in polyacrylamide gels, and increased after blotting to 2‐3%, whereas the detection efficiency for 35S was 2‐3% in polyacrylamide gels. An increase in gel thickness reduced the detection efficiency of the Linear Analyzer significantly, an effect which was also observed in fluorography. The instrument can be used for direct, quantitative evaluation of tritium in gels and blots because a linear relationship exists between the input radioactivity and the recorded counts. Double‐label experiments with 3H‐ and 35S‐labeled proteins can also be quantified. The spatial resolution of the Linear Analyzer reaches the quality attained in fluorography. Quantitative evaluation of gels and blots by the Linear Analyzer has the additional advantage of being less time‐consuming than detection by fluorography.
Analysis of human haptoglobin‐hemoglobin complexes by micro two‐dimensional electrophoresis
Authors: Manabe Takashi , Takahasi Yuko , Okuyama Tsuneo
Published: May 2005

A microscale‐multisample two‐dimensional electrophoretic technique, in which no denaturing agent is used, was applied to the analysis of three phenotypes of haptoglobin and their hemoglobin complexes in human sera. Haptoglobin‐hemoglobin complexes did not dissociate during the electrophoretic run, so their pIs and apparent molecular weights could be estimated. The number of spots of haptoglobin‐hemoglobin complex derived from a haptoglobin molecular species coincided with the number of β chains involved in it. Heterogeneity in pI of the haptoglobin‐hemoglobin complexes was observed, which suggests the heterogeneous binding of an αβ half‐molecule of hemoglobin to a β chain of haptoglobin.
Improved protein analysis on nitrocellulose membranes
Authors: Polvino William J. , Saravis Calvin A. , Sampson Christian E. , Cook Richard B.
Published: May 2005

The use of peroxidase‐conjugated antibodies for detecting specific tumor associated antigens bound to nitrocellulose membranes is being increasingly used in our laboratory. This method offers many advantages. However, we have observed several problems which can cause confusing results and possible misinterpretations. Some methods of eliminating artifacts are described as well as the use of glutaraldehyde vapor fixation as a means to increase sensitivity.
Data treatment in multichannel isotachophoresis
Authors: Schumacher Ernst , Arn Dieter , Thormann Wolfgang
Published: May 2005

This paper describes a microcomputer‐based method for collection and treatment of data from an analytical isotachophoresis apparatus having an array of detectors along the separation trough. The electric field distribution within the separation column is measured as a function of time by means of 256 equidistant detector electrodes. This allows monitoring the detailed approach of the steady isotachophoretic state in a fully automated fashion. Collecting data as well as software for data treatment are discussed together with illustrations.
Immobilized pH gradients for isoelectric focusing: Interaction between histones and histone‐like proteins with the charged polyacrylamide matrix
Authors: Righetti Pier Giorgio , Delpech Marc , Moisand Françoise , Kruh Jacques , Labie Dominique
Published: May 2005

It has been found that polyacrylamide matrices containing immobilized pH gradients interact strongly with at least two classes of proteins, histones and the histone‐like, ‘high‐mobility group’ (HMG) chromatin proteins, forming insoluble complexes. The nature of these interactions has been demonstrated to be purely ionical: the complexes are split by high ionic strength (0.5 M NaCl) and by altering the pH (full disaggregation being obtained at pH 5.5 and at pH 11.5). On the basis of these observations, soluble Immobiline polymers have been made, by polymerizing them in the absence of cross‐linker (N,N′‐methylenebisacrylamide). Several classes of Immobiline polymers can be produced: a) mixed‐type polymers, containing a blend of basic and acidic Immobilines, in such ratios as to obtain any desired pH in solution; b) homopolymers with a pure carboxyl surface; and c) homopolymers with a pure amino surface. These polymers can be synthesized as ‘mono‐pK species’ or as ‘multiple‐pKcoils’, the latter providing a full range of charges over an ample pH‐interval. It has been found that ‘carboxyl’ Immobiline homopolymers preferentially interact with the HMGl component among the HMG class, thus allowing the partial purification from a lysate by a simple precipitation step.
Detection of binding artifacts in gel electrofocusing using a polymeric dye probe
Authors: Cuono Charles B. , Chapo Giselle A. , Chrambach Andreas , Hjelmeland Leonard M.
Published: May 2005

Poly R‐480, an amphoteric dye of molecular weight 160 000,is the product of copolymerization reaction and can be expected to yield a polydisperse pattern in polyacrylamide gel isoelectric focusing (PAGIF). Such is indeed obtained in conventional PAGIF using commercial synthetic carrier ampholyte mixtures but not in buffer PAGIF, in which a few discrete dye bands are produced. Upon excision of single bands and segments of zones in buffer PAGIF and PAGIF in SCAMs, respectively, the entire original pattern is reestablished in each case, thereby confirming the artifactual nature of both the simple band pattern as well as the polydisperse patten. The simple pattern in buffer PAGIF prevails in the presence of 8 M urea and 8 mM CHAPS, while the polydisperse pattern obtained in SCAMs is resolved into multiple bands in the presence of either one or both of these agents. These observations suggest: a) hydrophobic and hydrogen bond dye‐dye and/or dye‐SCAM interactions produce the artifactual polydisperse pattern in PAGIF in SCAMs, b) electrostatic dye‐dye interactions are responsible for the artifactual simple band pattern in buffer EF, and c) dye‐SCAM electrostatic interactions produce the artifactual multiple band pattern in the SCAM‐urea‐CHAPS system. These data clearly underline the need for critical experimentation in the evaluation of PAGIF bands and band numbers.
Detection of α‐amylase isoenzymes by means of two‐dimensional electrophoresis followed by blue starch staining
Authors: Kadofuku Tsuyoki , Sato Tsuneo , Manabe Takashi , Okuyama Tsuneo
Published: May 2005

α‐Amylase isoenzymes present in human body fluids were separated by two‐dimensional electrophoresis in the absence of denaturing agents and detected by blue starch staining. Human body fluid samples (5 sera, 2 salivas, 1 pancreatic juice and 1 urine) were subjected to the analysis. Two to six activity spots of α‐amylase were detected for each sample. The spots were classified into four main species by their isoelectric points (pI) 6.9, 6.5, 5.8 and 5.3. All the α‐amylase isoenzymes showed apparent molecular weights above 140 000.
Staining‐destaining tube for polyacrylamide disc gels
Authors: Baumstark John S.
Published: May 2005

An 18 × 150 mm test tube in whose sides 3–5 mm holes have been blown is described. This tube(s) offers an economical alternative to commercially available staining‐destaining apparatus for polyacrylamide disc gels.
Electrophoretic extraction‐concentration of proteins from polyacrylamide gels under alkaline, neutral and acidic conditions
Authors: Hashizume Shuichi , Rashid Mohammad A. , Shoji Masahiro , Kuroda Kazuhiko
Published: May 2005

A simple technique has been developed for the electrophoretic extraction of milligram to microgram quantities of protein, under constant monitoring by dye line, in a concentrated form from polyacrylamide gels run under alkaline, neutral and acidic conditions. Typically, the extraction time from 5 gel slices per tube containing about 1.2 mg of bovine serum albumin or α‐chymotrypsinogen A was 3 h, and the maximum concentration of the recovered protein was 5 mg/ml with a total recovery of more than 90% in all cases. Higher protein concentrations (11 mg/ml) could be obtained by increasing the extraction time to 3.5 h or by using resins like glass beads. The alkaline extraction system has been applied to the purification of glutamyl‐tRNA synthetase up to homogeneity, and also to ferritin. This technique can also be used under reducing conditions and the scale of operation can be adjusted by the number of tubes or number of slices.
In vitro [35S]methionine‐labeled protein synthesis in microdissected discrete brain areas: Marked regional differences revealed by two‐dimensional gel electrophoresis
Authors: Gold Mark A. , Heydorn William E. , Creed G. Joseph , Weller Joan L. , Klein David C. , Jacobowitz David M.
Published: May 2005

A method is presented for the incorporation of [35S]methionine into proteins from freshly microdissected brain tissue samples. Rats were killed by decapitation, the brain was rapidly removed and sectioned without freezing using a vibratome. Hypothalamic nuclei and circumventricular organs of interest were removed by microdissection and incubated in a media containing [35S]methionine for six hours. Proteins within these tissue samples were then separated by two‐dimensional polyacrylamide gel electrophoresis. The gels were stained with silver, photographed, dried and exposed to autoradiographic film. The results suggest that 1) this technique will prove useful in examining the uptake of radiolabeled amino acids into proteins from discrete nuclei and other brain areas, and 2) there is considerable regional heterogeneity in the uptake of [35S]methionine by proteins from the different brain regions examined in this study.
Forming reproducible density and solute gradients by computer‐controlled cooperation of stepmotor‐driven burettes
Authors: Altland Klaus , Altland Alexander
Published: May 2005

A program has been developed for the computer‐controlled cooperation of 4 commercially available stepmotor‐driven burettes to form density gradients in polyacrylamide gels and solute gradients for other purposes such as centrifugation and low pressure chromatography. The slope of the gradients was defined by entering up to 200 endpoints of straight lines with the percentage of the total volume and the concentration of the gradient‐forming solute as coordinates. The possible minimum increment of the discontinuous gradients is 0.5 % of the total range. They are mixed at variable flow rate by two burettes containing the high and low density acrylamide solutions and two additional burettes for the admixture of two catalysts at variable volume proportions. Within 10 min gradients with total volumes between 4 and 650 ml can be delivered. Gradient volumes smaller than 4 ml are possible if an average increment of > 0.5 % is accepted. The program meets the requirements of reproducibility for high resolution gradient gel electrophoresis and isoelectric focusing in immobilized pH gradients. All input data for the definition of the gradients can be displayed as a hard copy by a matrix printer and stored on tape. Thus the device meets the requirements of proper documentation of a given gradient in a publication and for reproduction in another laboratory. Other types of gradients for volumes of up to 1.4 liters can be produced by a subprogram converting the 4 burettes into a gradient pump with continuous outflow as used for column chromatography. The speed is variable between 0.4 ml and 5.2 liters per hour with minor pulsation only in the very low range.
A preliminary evaluation of typing commonly occurring human erythrocyte acid phosphatase (ACP1) phenotypes in bloodstains, using the LKB Immobiline system
Authors: Westwood Sara A. , Sutton John G.
Published: May 2005

A preliminary study has been made of the use of immobilised pH gradients (Immobiline gels) for the separation of the six commonly occurring erythrocyte acid phosphatase (ACP1) phenotypes in blood lysates and stains. These pH gradients are immobilised in the matrix of the polyacrylamide gel and possess different properties from conventional isoelectric focusing (IEF) gels. A comparison was made between the profile of the ACP1 isoenzymes apparent on conventional IEF gels with that seen on the Immobiline gels.
Fish species identification by electrophoresis of muscle proteins in sodium dodecyl sulfate‐containing polyacrylamide slab gels
Authors: Corzo Javier , Riol‐Cimas José M. , Meléndez‐Hevia Enrique
Published: May 2005

Fish muscle proteins were separated by slab gel electrophoresis in 10–20 % polyacrylamide gradient gels in presence of sodium dodecyl sulfate. Three muscle extracts were tested: whole homogenate, sarcoplasmic and myofibrillar. Based on differences of the patterns in the 14 000–205 000 molecular weight range, identification of species of different orders, families and genus seems feasible. The most reproducible patterns were obtained with the whole homogenate. No changes were detected in the patterns after cold storage for several days (without freezing).
Calculation of “Tris‐borate” ion mobiliy
Authors: Michov Budin M.
Published: May 2005

The mobility of the “Tris‐borate” ion at different temperatures and ionic strengths is calculated using previously published equations for calculating the approximate mobility of composed ions.