bluuebunny/crossref_metadata_2025 huggingface dataset:
Ferguson plots based on absolute mobilities in polyacrylamide gel electrophoresis: Dependence of linearity of polymerization conditions and application to the determination of free mobility
Authors: Butterman Michael , Tietz Dietmar , Orbán László , Chrambach Andreas
Published: May 2005

In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacrylamide gel electrophoresis based on their absolute mobilities were found to be linear under unusual polymerization conditions which yield relatively wide gel fibers and a low total fiber length per unit weight, but not under previously and commonly used conditions. These linear Ferguson plots in gels of 1, 3 and 5% crosslinking intersect at a single gel concentration between 1 and 2 %T (M‐point). It is postulated that the measure of free mobility of the proteins is the M‐point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously. This postulate abolishes the well‐known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo) in terms of free mobility. The postulate is also compatible with the interpretation of the points of intersection of the Ferguson plots of oligomeric series of proteins at finite gel concentrations (designated μ‐points) as their common free mobilities.
The use of biotinylated DNA probes in parentage testing: Non‐isotopic labeling and non‐toxic extraction
Authors: Dykes Dale D.
Published: May 2005

With a few exceptions DNA probing techniques require the use of radioisotopes and toxic DNA extraction techniques which render the method expensive, potentially hazardous and time‐consuming. Most isotopic labeling techniques use the isotope 32P and require 3–10 days to visualize bands after hybridization. An alternative approach is based on the use of non‐isotopic detection methods. The available nonisotopic techniques were assessed and their practicality tested. All probes analyzed were tested on samples extracted with a non‐toxic extraction procedure using 6 M NaCl as the substitute for phenol and isochloroform. By manipulating probe sizes, blocking agents, selection of membrane and detection system, it is feasible to use non‐isotopic labeling and detection in routine parentage testing. Reproducible results were obtained with labeling a variety of DNA probes of various sizes, plasmids and inserts. With an absence of waste disposal costs, probes that are stable for over two years and a staining procedure which takes 3–5 h versus days the technique is well suited for a normal laboratory setting. The next key to the acceptability of DNA testing will be the commercial availability of DNA probes for widespread use.
Application of plasminogen polymorphism to forensic hemogenetics
Authors: Skoda Ulrike , Klein Anne , Lübcke Ingeborg , Mauff Gottfried , Pulverer Gerhard
Published: May 2005

Plasminogen polymorphism (PLG) has attained considerable importance in forensic hemogenetics. PLG comprises two common, codominant autosomal alleles, PLG*A and PLG*B, more than 18 variants, and the silent allele PLG*QO. Isoelectric focusing followed by functional or immunochemical detection seems to be the optimal method for the determination of phenotypes. PLG*A is the most common allele in all populations, having its highest frequency in Mongoloids, Amerindians and Eskimos, the lowest in Caucasoids. The functionally inactive plasminogen M5 so far has been seen exclusively in Japanese individuals. Silent PLG alleles were only observe in the heterozygous state. No clear differences in functional activity or plasma level could be ascertained for any of the other allotypes. PLG polymorphism is now widely used for many haemogenetic investigations. From the allele distribution in European Caucasoids a single exclusion chance of 17.2 % for non‐fathers in paternity testing may be calculated. The major prerequisites of a new genetic marker in the parentage expertise, established Mendelian inheritance, favorable distribution of common alleles, low frequency of silent alleles, and simple reproducible typing technology, are fulfilled.
Subtyping of phosphoglucomutase 1 (PGM1) in microstains of different biological materials with subsequent species differentiation without further stain material consumption
Authors: Pflug Werner
Published: May 2005

Micromethods for subtyping of phosphoglucomutase 1 (PGM 1) in small amounts of biological stain material are described, using an applicator for highly diluted stain extracts. With the aid of this applicator strip blood and semen micro‐stains as well as single hair‐roots could be extracted by electroelution directly on the PGM 1 isoelectric focusing gel. Species differentiation was also possible either by radial immunodiffusion using the extract remaining in the applicator strip after isoelectric focusing or by interpretation of the PGM pattern itself.
Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients
Authors: Kinzkofer‐Peresch Angelika , Patestos Nikos P. , Fauth Markus , Kögel Friedrich , Zok Roland , Radola Bertold J.
Published: May 2005

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5 %T, 3 %C, were internally stabilized with the aid of an AcrylAide‐pretreated, hydrophilized polyester fabric, preferably with a 60 μm mesh opening. Ultrathin (120–180 μm) gels were prepared with the flap technique and 500 μm gels with the cassette technique; 500 μm gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1–3 % w/v carrier ampholytes. Incorporation of 40–60 % w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900–1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1–0.2 μm pore size nitrocellulose membranes in 10–20 min from ultrathin gels and in 30–60 min from 500 μm gels. Based on quantification of residual protein in the gels after blottinǵ, a transfer efficiency of 60–87 % was found for the ultrathin and 53–69 % for the 500 μm gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris‐glycine buffer system electrophoretic blotting required only 2–5 min with ultrathin gels and 20 min with 500 μm gels. Marker proteins, including horse spleen ferritin (Mr465 000), could be transferred with 91–96 % efficiency.
Immunochemical detection of peptides and proteins on press‐blots after direct tissue gel isoelectric focusing
Authors: van der Sluis Paul J. , Pool Chris W. , Sluiter Arja A.
Published: May 2005

A sensitive method is described for the detection of tissue peptides and proteins. They are separated by tissue isoelectric focusing using thin large‐pore polyacrylamide gels, containing detergent and dimethylformamide, and are fixed with either glutaraldehyde or formaldehyde in gelatin‐coated nitrocellulose membranes using press‐blotting. The fixed peptide and protein antigens are visualized by immunoperoxidase staining. The spectrum of fixed tissue constituents may also be used to test antiserum reactivity and specificity in immunocytochemical staining procedures. Isoelectric focusing of 2 μL homogenates of the neurointermediate lobe of the pituitary allowed the immunodetection of peptides and proteins of various sizes and the determination of isoelectric points. However, direct application onto gels of small pieces of frozen tissue sections, sliced in a cryostat, appeared to be more efficient. By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 μm thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.
Extraction of plant proteins for two‐dimensional electrophoresis
Authors: Granier Fabienne
Published: May 2005

Three different extraction procedures for two‐dimensional electrophoresis of plant proteins are compared: (i) extraction of soluble proteins with a nondenaturing Trisbuffer, (ii) denaturing extraction in presence of sodium dodecyl sulfate at elevated temperature allowing the solubilization of membrane proteins in addition to a recovery of soluble proteins, and (iii) a trichloroacetic acid‐acetone procedure allowing the direct precipitation of total proteins.
Two‐dimensional electrophoresis of cereal prolamins: Applications to biochemical and genetic analyses
Authors: Shewry Peter R. , Parmar Saroj , Field J. Michael
Published: May 2005

Three complementary two‐dimensional systems for the analysis of cereal prolamins are described. These are electrophoresis at pH 3.1 followed by electrophoresis at pH 9.2, isoelectric focusing (IEF) followed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and SDS‐PAGE under non‐reducing conditions followed by SDS‐PAGE with reduction. They together give information on the pIs, Mrs and charge properties of the individual prolamin components, and on their interactions to form oligomers stabilized by inter‐chain disulphide bonds. The three systems are then compared for their effectiveness in analysing unreduced prolamin I fractions from wheat and rye, and the IEF/SDS‐PAGE system for analysing reduced and pyridylethylated prolamin fractions from all the major cereals. Finally, applications of the systems in biochemical and genetic studies are discussed and illustrated with three examples: analysis of the structural relationships of the S‐rich prolamins (B hordeins and γ‐type hordeins) of barley, determination of the interactions of wheat and rye prolamin subunits in a 2RS/2BL translocation line, and the mapping of genes for α‐type prolamins in the wild grass Haynaldia villosa.
Electrophoretic heterogeneity of maize histones
Authors: Ivanchenko Maria G. , Georgieva Elena I.
Published: May 2005

Histones from maize embryos and seedlings have been isolated using a fast extraction procedure. Three different electrophoretic systems have been applied for the study of the heterogeneity of maize core histones. Electrophoresis in acetic acid/urea polyacrylamide gels, containing high concentrations of urea, resulted in optimum fractionation of the core histones and especially of histone H4. Sodium dodecyl sulfate‐ containing polyacrylamide gels were not useful for the fractionation of maize histone classes H2a and H2b, nor for the various subfractions of H3 and H4. Gels containing Triton X‐100, used for the dimension in two‐dimensional electrophoresis proved to be efficient for the separation of all histone classes, as well as their structural variants and chemical modifications. Maize core histones have been oxidized in an attempt to define which of the Triton X‐100 resolved subfractions represent oxidation forms.
Routine diagnosis with PhastSystem compared to conventional electrophoresis: Automated sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, silver staining and Western blotting of urinary proteins
Authors: Scherberich Jürgen E. , Fischer Peter , Bigalke Annette , Stangl Pia , Wolf Gunter B. , Haimerl Marianne , Schoeppe Wilhelm
Published: May 2005

The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient‐gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis (SDS‐PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional “macro”‐method with self‐cast SDS‐polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 μL sample volumes and an 8–25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS‐PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha‐1‐antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS‐PAGE and staining allows easy standarization of urine SDS‐PAGE among clinical routine laboratories.
Two‐dimensional electrophoretic analysis of human milk proteins
Authors: Goldfarb Marcia F. , Savadove Maureen S. , Inman Jean A.
Published: May 2005

Identification of human milk proteins and formulation of a two‐dimensional map is a first step in a project which intends to survey human milk proteins by two‐dimensional electrophoresis. Thirty‐four proteins have been identified using the Iso‐Dalt method of separation and Western blot with immunoprobes. Identification confirms that milk is species‐specific, and, therefore, breast feeding confers a decided advantage for the infant. Using antisera for identification has revealed relationships between molecules which have not been previously noted. The antibody recognizes a common epitope between the IgA α chain and lactoferrin, and between the IgD d chain and β casein. Milk protein concentrations vary longitudinally, diurnally, and individually. Identification of the proteins contributes meaning to the varying patterns. Knowledge of human milk proteins will help to elucidate human nutrition and health needs.
Free flow electrophoresis as a method for the purification of enzymes from E. coli cell extract
Authors: Kuhn Reinhard , Wagner Horst
Published: May 2005

The application of the four techniques of free flow electrophoresis (zone electrophoresis, isotachophoresis, isoelectric focusing and field step electrophoresis) for the purification of proteins from a complex protein mixture was investigated. For this purpose α‐amylase (EC 3.2.1.1) from Aspergillus oryzae was added and reisolated from E. coli cell extract. The chosen enzyme and the biological extract are models for many industrial separation problems. In optimized experiments purity, purification factor, yield, throughput and efficiency were calculated. The best results were obtained with field step electrophoresis in combination with zone electrophoresis. High purity (0.82 mg enzyme/mg total protein) and high throughput (111 mL sample/h) were achieved using this technique. Field step electrophoresis gave the best throughput (330 mL sample/h), but low purity (0.63 mg enzyme/mg total protein). This technique can also be used for a simple concentration of the sample. With zone electrophoresis a purity of more than 0.95 mg enzyme/mg total protein was obtained, which was the best of all techniques. However, the enzyme concentration was decreased due to dilution with buffer solution after the separation. Isotachophoresis was the most difficult technique, combined with a relatively low recovery of 31% of the enzyme activity. In a purification scheme, free flow electrophoresis is able to substitute one or even several chromatography steps with a negligible loss of biological activity.
The microheterogeneity of serum α1‐antichymotrypsin revealed by interaction with concanavalin A in crossed immunoaffinoelectrophoresis and in affinity chromatography
Authors: Laine Anne , Hachulla Eric , Hayem Annette
Published: May 2005

In crossed immunoaffinoelectrophoresis with free concanavalin A in the first dimension, human serum α1‐antichymotrypsin, purified or in whole serum, exhibited four peaks in presence of 0.02 M α‐methyl‐D‐glucoside added to the second‐dimensional gel. α1‐Antichymotrypsin purified from the serum of a single healthy donor was separated by affinity chromatography into three fractions on a laboratory‐prepared concanavalin A‐Sepharose 4B column: a pass‐through fraction, a retarded fraction and a bound fraction, eluted from the column on addition of sugar to the buffer. These three fractions were analyzed by crossed immunoaffinoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and isoelectric focusing before and after desialylation. The results of electrophoresis as well as chemical analyses indicate that these microheterogeneous forms carry glycans with decreasing degrees of branching from the concanavalin A‐pass‐through form to the concanavalin A‐bound form. This approach represents a first step towards the elucidation of the molecular basis of the microheterogeneity of α1‐antichymotrypsin.
Study of human cerebrospinal fluid proteins by size exclusion‐high performance liquid chromatography and two‐dimensional gel electrophoresis
Authors: Wiederkehr Felix , Büeler Martin R. , Wacker Marianne , Vonderschmitt Dieter J.
Published: May 2005

Cerebrospinal fluid (CSF) proteins were separated into three main fractions by size exclusion‐high performance liquid chromatography (SE‐HPLC). Subsequent analysis of each fraction by two‐dimensional gel electrophoresis (2‐DE) facilitated the detection of trace components in CSF and additionally provided more information about the native properties of various proteins. Certain proteins are present in a polymeric form and appear in the high molecular weight SE‐HPLC fraction. In the middle molecular weight SE‐HPLC fraction we found a CSF‐specific transthyretinrelated protein by immunoblotting with polyclonal antibodies to transthyretin. Possible interpolypeptide disulfide bonds of such polymeric proteins were studied using a nonreducing 2‐DE system. This procedure revealed that all apolipoprotein E monomers in CSF, which are synthesized in astrocytes, are linked by disulfide bonds. In the CSF from a patient with clinically definite multiple sclerosis (MS), novel proteins appeared in the high molecular weight SE‐HPLC fraction, which are obscured by other proteins if total CSF is analyzed.
Detection of β‐ 1,3‐glucanase activity after native polyacrylamide gel electrophoresis: Application to tobacco pathogenesis‐related proteins
Authors: Côté Francois , Letarte Jocelyne , Grenier Jean , Trudel Jean , Asselin Alain
Published: May 2005

β‐1,3‐Glucanase (laminarinase) activity was detected after polyacrylamide gel electrophoresis under native conditions by using laminarin as substrate. Following incubation of gels, laminarin was stained with Aniline Blue. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. As low as 0.001 unit of commercial Penicillium laminarinase could be observed after incubating the polyacrylamide gel for 45 min at pH 5.0. Extracts of commercial Penicillium laminarinase exhibited four bands with lytic activity towards laminarin. Analysis of intercellular fluid extracts of tobacco mosaic virus‐infected tobacco leaves revealed four β‐1,3‐glucanases corresponding to three acidic pathogenesis‐related proteins, b4 (2), b5 (N) and b6b (0), and one basic protein. The presence of laminarin in gels retarded the migration of some proteins with β1, 3‐glucanase activity. This change in electrophoretic mobility could be used as a complementary affinity test for identifying proteins with β‐1,3‐glucanase activity.
Affinity electrophoresis for diagnosis fo cancer and inflammatory conditions
Authors: Brȩlorowicz Jan , Mackiewicz Andrzej
Published: May 2005

Affinity electrophoresis, with concanavalin A and Lens culinaris agglutinin as ligands, was applied to study the microheterogeneity of serum proteins, with special emphasis on α1‐fetoprotein and α1‐acid glycoprotein, two proteins of potential clinical value. A total of 602 samples of serum from patients with various neoplastic and inflammatory conditions were evaluated. Affinity electrophoresis provided useful information for differential diagnosis of cancers of various origins including hepatomas, metastatic liver cancers and yolk sac tumors. The method also proved indispensable for the detection of intercurrent infection in patients with rheumatoid arthritis, systemic lupus erythematosus and scaled burns.
Two‐Dimensional electrophoresis of human serum proteins following acute myocardial infarction
Authors: Marshall Thomas , Williams John , Williams Katherine M.
Published: May 2005

Serum proteins associated with acute myocardial infarction (AMI) have been monitored by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and high resolution two‐dimensional electrophoresis (2‐DE) under nonreducing conditions. Proteins a, b, c (Mr 13 000; pI 6.2, 6.7 and 7.5, respectively) and e (Mr 27 000; pI 5.2) appear simultaneously ∼ 30 h after infarction, reach maximum intensity after 48 h and progressively decline thereafter. Protein d (Mr 15 000; pI 7–8.5; identified as hemoglobin) sometimes appears within 18 h of infarction. Proteins a‐c are not detected in the 2‐DE patterns of healthy myocardium, infarcted myocardium, pectoral muscle or tongue, but e is present in all and tentatively identified as myosin light chain. Other myocardial proteins which are either reduced in amount following infarction or more specifically associated with myocardium than pectoral muscle are not detected in the serum of AMI patients. Analysis of unconcentrated urine by SDS‐PAGE and silver staining does not reveal proteins specific to AMI.
The impact of boundary conditions in free flow field step electrophoresis
Authors: Heydt Ariane , Mosher Richard A.
Published: May 2005

The impact of the type of membrane used to separate the electrolyte compartments from the separation cell for continuous flow field step electrophoresis is examined using both experimental and computer simulation data. It is shown that the distinct transport characteristics of ion exchange membranes and dialysis membranes may have an effect on the stability of the pH and conductivity gradients created by the buffer system. These effects are interpreted using computer simulation data. The limitations of the model used to portray the transport characteristics of ion exchange membranes are discussed as are the experimental conditions which provide the greatest stability for field step electrophoresis.
The electric field dependence of DNA mobilities in agarose gels: A reinvestigation
Authors: Holmes Diana L. , Stellwagen Nancy C.
Published: May 2005

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments ≤ 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained ≤ 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris‐borate buffer (TBE) than in gels cast and run in Tris‐acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incroporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature‐corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments ≤ 12 kbp in size in agarose gels ≤ 1.4 % in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments ≤ 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments ≥ 1 kbp in size in agarose gels ≥ 2 % in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an “intrinsic” DNA mobility of 2.7 x 10−4 cm2/Vs at 20°C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments. If the volume occupied by each DNA molecule is approximated by its root‐mean‐square (rms) radius of gyration, the median pore radius, rp, of an LE agarose gel cast and run in TAE buffer is rp = 98 A−0.90, where A is the agarose concentration in % and rp is given in nm. For gels cast and run in TBE buffer, the apparent median pore radius is rp = 66 A−0.95. If the volume occupied by each DNA molecule is represented by its mean geometric radius instead of the rms radius of gyration, the median gel pore radius in both buffers depends on A−0.5, as predicted by the extended Ogston theory. From the corresponding retardation coefficients, the gel fiber parameters were estimated.
Rapid detection of proteins by enzyme‐linked immunofiltration assay after transfer onto nitrocellulose membranes
Authors: Pinon Jean‐Michel , Puygauthier‐Toubas Dominique , Lepan Hervé , Marx Cathy , Bonhomme Annie , Boulant Joselyne , Geers RÉGine , Dupont Hervé
Published: May 2005

Enzyme‐linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 μm). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetetive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1–5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.
A procedure for the extraction and high resolution two‐dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei
Authors: Baciu Peter C. , Durham John P.
Published: May 2005

Methods are described for the extraction and preparation of total nuclear proteins for high resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post‐translational modifications. Unlike other procedures for 2‐D PAGE analysis of nuclear proteins the technique allows solublization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi‐labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2‐D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2‐D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.
Giant two‐dimensional gel electrophoresis: Methodological update and comparison with intermediate‐format gel systems
Authors: Levenson Richard M. , Anderson George M. , Cohn Jonathan A. , Blackshear Perry J.
Published: May 2005

Two‐dimensional (2‐D) gel electrophoresis methods for separating complex mixtures of proteins have not changed fundamentally since their original description in the late 1970′s. Nevertheless, 2‐D gel resolution has improved substantially as a result of a series of incremental modifications. One of these was the development of the „giantgel”︁ format, using gels measuring at least 30 x 30 cm to provide the highest resolution 2‐D gel system available. As originally described, this procedure has several important limitations: it requires custom‐built equipment; it is expensive in terms of time, reagents, film and support matrices; and it generates gels which are difficult to manupulate, particularly for silver staining. This report describes modifications in the giant gel procedure to permit use of a commercially available gel apparatus and to obtain gaint gels of improved mechanical strength suitable for silver staining. The resolution of giant gels is compared with that obtained using two systems currently being marketed for use by laboratories performing large numbers of 2‐D gel analyses. The smaller format gels resolved fewer proteins, by 30–40%, compared with the giant gels. This difference in resolving power suggests that giant gels will continue to be useful in selected applications.
A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis
Authors: Ali Rashda , Sayeed Syed Asad
Published: May 2005

A reddish‐brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11–12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2–2h with 7% acetic acid at 60°C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R‐250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity.
Electrophoresis and densitometry of serum and urine in the investigation and significance of monoclonal immunoglobulins
Authors: Macnamara Elizabeth M. , Whicher John T.
Published: May 2005

About 15% of monoclonal components or paraproteins are associated with malignancy when detected by simple electrophoresis on cellulose acetate membranes or agarose gel followed by protein staining. More sensitive methods for detecting monoclonal components result in a lower frequency of association with recognisable underlying disease. The sensitivity is dependent on the system used. Isoelectric focusing is 10 to 40 times more sensitive than simple electrophoresis at detecting monoclonal components. Electrophoretically separated bands may be quantitated densitometrically and at the present time this is the most satisfactory method for measuring monoclonal component concentration. Immunochemical methods for quantitating monoclonal components are limited by failure to react in a similar manner to polyclonal standards and giving problems of antigen excess resulting both in falsely elevated and low results. Electrophoretic methods must always be used in conjunction with these. The normal polyclonal ratio of immunoglobulin kappa:lambda light chain is distrubed by the presence of a monoclonal component. In 260 patients with myeloma we found that serum electrophoresis alone detected 90% while a disturbance in the kappa:lambda ratio detected 98%. However, 50 % of monoclonal components less than 3 g/L were missed. Log kappa:lambda ratio correlates well with the densitometric scan of monoclonal components, both in vitro and in patients being monitored for treatment response in myeloma. This approach may complement or even replace densitometry for this purpose in the future.
Two‐dimensional gel electrophoresis, protein electroblotting and microsequencing: A direct link between proteins and genes
Authors: Bauw Guy , Rasmussen Hanne Holm , Bulcke Marc Van Den , Damme Jozef Van , Puype Magda , Gesser Borbala , Celis Julio E. , Vandekerckhove Joël
Published: May 2005

We have used two‐dimensional gel electrophoresis as a general „preparative”︁ method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract of Nicotiana tabacum leaf tissue were directly blotted from the gel onto poly(4‐vinyl‐N‐methylpyridinium iodide)‐coated glass fiber sheets. The major spots were excised and subjected to NH2‐terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two‐dimensional gels and the Coomassie Brilliant Blue‐stained spots were excised from the gels. The combined spots were re‐eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented by in situ proteolysis and the generated peptides were separated by reverse phase–high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two‐dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or forthcoming DNA sequence data.
Separ of cationic proteins via charge reversal in capillary electrophoresis
Authors: Wiktorowicz John E. , Colburn Joel C.
Published: May 2005

Analysis of proteins by capillary electrophoresis requires strategies which minimize coulombic interactions with the capillary surface. Thus buffers with pH's above the isoelectric points (pI) of proteins, or near the pI of silanol are required for efficient separation. Covalent modification of the capillary surface is also effective; however, this strategy is technically difficult, abolishes endosmotic flow and suffers from the inherent lability of the siloxane bond. Finally, „dynamic coating”︁ agents, which interact weakly with the capillary surface and therefore, must be included in the separation buffer, suffer from the potential interaction of coating agent with analytes, altering the selectivity of the system. In the following paper, we describe another approach which overcomes all of these difficulties, and demonstrate the ease of use, nondenaturing property, stability and selectivity of the coating strategy with several model protein systems.
Monocyte‐specific esterase isoenzyme demonstrated by isoelectric focusing
Authors: Gignac Suzanne M. , Drexler Hans G.
Published: May 2005

Using horizontal thin‐layer isoelectric focusing in polyacrylamide gels, we separated the isoenzymes of carboxylic esterase (EC 3.1.1.1) of cell extracts prepared from human hematopoietic cells. Isoenzyme bands were visualized by staining with naphthol ester as substrate and coupling to an azo dye. Staining intensities of isoenzymes were quantified by densitometric scanning. On isoelectric focusing in a pH 2–11 gradient, distinct esterase isoenzyme profiles could be discerned and correlated to various types of normal hematopoietic cells and their leukemic counterparts. One unique isoenzyme, termed monoband, could be clearly identified on the basis of its isoelectric point (pI 6.0), its strong expression by normal and malignant monocytes and its complete and selective inhibition by sodium fluoride. This band was only found in monocytes of either normal or leukemic origin, but not in lymphoid or myeloid cells. The monocyte esterase could be inhibited by sodium fluoride whereas other isoenzyme bands were resistant to this inhibition. However, the specificity of this inhibitory reaction was relative, depending on the concentration of sodium fluoride. Compared with normal monocytes, leukemic monocytes often showed an overexpression of the mono‐bands. Dilution experiments established the distinct prominence of the mono‐band which could be detected among the other isoenzymes even when only 1% of the total cell population consisted of monocytes. Immature myeloid, but mono‐band negative leukemic cells whose arrest of differentiation can be overcome by in vitro 12‐O‐tetradecanoylphorbol 13‐acetate‐promoted differentiation to more mature cells, could be induced to express the mono‐band which paralleled their maturation to monocytes. Isoelectric focusing of esterase isoenzymes represents a powerful tool for the isoenzymatic analysis of hematopoietic cells and an enzymological classification of leukemias.
Electrophoretic characterization of posttranslational modifications of human parotid salivary α‐amylase
Authors: Bank Ruud A. , Hettema Ewald H. , Arwert Fré , Amerongen Arie V. Nieuw , Pronk Jan C.
Published: May 2005

Human salivary α‐amylase displays multiple bands upon native polyacrylamide gel electrophoresis. In fresh saliva, due to posttranslational modifications, a pattern of 5–6 isozymes is observed. The isozymes are designated 1–6, in the order of increasing anodal mobilty. As a result of the development of a rapid and sensitive electrophoresis system, with markedly higher resolution than previously reported, we concluded that a previously proposed model (Karn et al., Biochem. Genet. 1973, 10, 341–350) is inadequate to explain the origin of the various bands. We propose an alternative model that fits in with our new and previously made observations. According to this model, band 2 is the primary gene product and band 1 is its glycosylated counterpart with only one neutral oligosaccharide present on each molecule. Band 3 originates from band 1 by the transialidase‐catalyzed incorporation of sialic acid into the biantennary chain. Bands 4 and 6 originate from bands 2 and 4, respectively, by deamidation; band 5 is the deamidation product of amylase with an acidic oligosaccharide (band 3). Only a minor part of band 3 consists of the deamidation product of band 1. Peptide Asn‐Gly‐Ser (residues 427–429) is the most probable candidate for glycosylation; literature data suggests that deamidation occurs in the stretch Glu‐Asn‐Gly‐Lys‐Asp (residues 364–368) and Asn‐Gly‐Asn‐Cys (residues 474–477). Both glycosylation and deamidation might play a role in the clearance of amylase from the systemic circulation. The elecroporesis system described is a powerful tool to determine amylase isozyme distributions in health and disease, especially for the screening of alterations seen in ectopically produced amylase.
Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments
Authors: Orbán László , Chrambach Andreas
Published: May 2005

DNA fragments up to 9 kb in size were stacked and separated by polyacrylamid gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis using a discontinuous buffer system. Polyacrylamide gel at pH 8.9, 2°C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 μg/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72–23130 bp, while agarose gels at pH 8.5, 25°C, provided well‐resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 μg/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 × TBE (Tris‐borate‐EDTA) buffer, while that successfully applied to polyacrylamide is seven‐fold less than the ionic strength of conventionally used 1 × TBE buffer, with is substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification Rf, the migration distance relative to a sharply defined “buffer front” (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico‐chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened. (iii) The degree of dilution of the DNA sample becomes unimportant.
Des‐gamma‐carboxyprothrombin detection by immunoblotting after polyacrylamide gel affinoelectrophoresis in human plasmas
Authors: Belle Michèle , Hanss Michel , Guillaumont Mark , Leclercq Michel , Guinet Roland
Published: May 2005

In the absence of vitamin K or in the presence of the vitamin K antagonists, abnormal nonfunctional forms of prothrombin circulate in the blood. A reliable and reproducible technique, derived from traditional crossed affinoimmunoelectrophoresis in presence of calcium lactate, was developed and optimized. The technique is based on nondenaturing polyacrylamide gel affinoelectrophoresis, with calcium lactate, of plasma samples, followed by immunoblotting with rabbit anti‐human prothrombin serum and detection with an anti‐rabbit immunoglobulin peroxidase conjugate. Depending on the plasmas, one or two bands were visualized and quantified by densitometry of the immunoblots. The technique was able detect abnormal des‐gamma‐carboxylated prothrombins at concentration of 0.1 μg/mL.
Barley cultivar discrimination: II. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and isoelectric focusing with immobilized pH gradients
Authors: Weiss Walter , Postel Wilhelm , Görg Angelika
Published: May 2005

Isoelectric focusing performed with immobilized pH gradients was found superior to other commonly used electrophoretic methods for discrimination of 55 European winter and spring barley cultivars. Hordeins, the alcohol‐soluble proteins, yielded 32 different patterns, allowing identification of 22 cultivars and classification of the remaining ones into ten groups of two to eight cultivars each. Only 21 different hordein patterns were observed using horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by silver staining. Twelve cultivars exhibited unique hordein patterns, the remaining nine groups contained 2–11 cultivars. Resolution of isoelectric focusing with immobilized pH gradients was further enhanced in some cases when the patterns of urea/dithiothreitol‐soluble proteins were used instead of the hordein patterns. However, evaluation was more complicated because of the larger number of protein bands detected.
Evaluation of membranes used for electroblotting of proteins for direct automated microsequencing
Authors: Baker Cathy S. , Dunn Michael J. , Yacoub Magdi H.
Published: May 2005

Protein purification and characterisation have been age‐old problems for the biochemist. A new era has arisen with the advent of one‐ and two‐dimensional gel electrophoresis and high sensitivity automated protein microsequencing. These two tools along with electroblotting have made it possible to separate and analyse complex protein mixtures. We studied six different membranes compatible with Edman degradation chemistry to determine their efficiencies at binding proteins electroblotted from one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Their overall blotting‐sequencing properties were also evaluated. We found that the polyvinylidene difluoride‐based membranes out‐performed the glass‐based and polypropylene‐based membranes under our selec ted experimental conditions. The problems associated with electroblotting and microsequencing are discussed.
Identification of human esterase D subunits from the homodimeric and heterodimeric forms of five phenotypes by a new two‐dimensional isoelectric focusing method
Authors: Alonso Antonio , Visedo Guillermo , Sancho Manuel , Fernández‐Piqueras José
Published: May 2005

A new two‐dimensional isoelectric focusing method was developed to identify the human esterase D (EsD) subunits from the homodimeric and heterodimeric forms of five EsD phenotypes. EsD polymorphism was also analyzed by one‐diniensional isoelectric focusing under reducing and mild denaturing conditions to study the influence of dithiothreitol and low concentrations of urea on the focusing pattern of the EsD dimers.
On the relationship of amino acid composition to silver staining of proteins in electrophoresis gels: II. Peptide sequence analysis
Authors: Gersten Douglas M. , Rodriguez Lewis V. , George David G. , Johnston Dennis A. , Zapolski Edward J.
Published: May 2005

The quantification of proteins in silver‐stained electrophoresis gels has been limited by the differences in “stainability” of different proteins. Despite efforts by many researchers, the precise basis of the reaction between silver reagents and polypeptides is still unclear, and, depending on the formulation, may even differ. We have tested the hypothesis that differences in stainability among proteins can be attributed to diferences in di‐ or tripeptide composition. The results indicate that some order of protein structure other than short peptides accounts for the staining differences observed.
A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis
Authors: De Bolle Miguel F. C. , Goderis Inge J. , Terras Franky R. G. , Cammue Bruno P. A. , Broekaert Willem F.
Published: May 2005

A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. α‐purothionin, Urtica dioica agglutinin, and tobacco chitinase.
Alteration in the electrophoretic mobility of OmpC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Lobos Sergio R. , Mora Guido C.
Published: May 2005

Studies on Salmonella typhi and Salmonella typhimurium outer membrane proteins have shown that the relative position of OmpC porin in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis undergoes an important shift when the concentration of ammonium persulfate in the running gel is increased from 6 to 12 mM. The apparent molecular mass at these concentrations was estimated to be 34 and 40 kDa, respectively. Under similar electrophoretic conditions the apparent molecular mass estimated for OmpF was 37.6 an 38.2 kDa. Therefore, OmpC moves from a leading position to a position behind OmpF. For Escherichia coli OmpC the shift observed is less pronounced than that occurring in Salmonellae.