bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies to establish a reference pattern of slime‐forming, encapsulated Streptococcus cremoris plasma membrane antigens by crossed immunoelectrophoresis
Authors: Niskasaari Kaarina , Valkonen Kaija H. , Forsén Raili
Published: May 2005

The antigenic composition of the plasma membrane of the slime‐forming, encapsulated strain Streptococcus cremoris T5, originally isolated from the Finnish ropy sour milk product “viili”, was studied by crossed immunoelectrophoresis (CIE). Membrane proteins were solubilized with 1% Triton X‐100 extraction and mild sonication at pH 8.7. The solubilizing effect for membrane protein was investigated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Only quantitative differences were observed in the SDS‐PAGE patterns of the Triton X‐100 extracts as compared with that of the total‐protein sample. The anti‐membrane antiserum pool was collected over twelve months. The conditions for CIE were standardized using different antigen and antibody concentrations. The immunoprecipitation pattern with standard antisera was reproducible with separate membrane preparations. The solubilized membrane antigens could be stored at ‐20°C for two months with no changes in the precipitation pattern. A reference immunoprecipitation pattern with 19 precipitation lines was established for S. cremoris T5 membrane antigens. Two immunoprecipitates had a strong staining intensity with Coomassie Brilliant Blue, and one of these was also stainable with periodate‐Schiff's reagent. Certain special characteristics of these precipitation lines are proposed for further study.
Detection of DNA and lipopolysaccharide in thick polyacrylamide gels using an improved silver stain
Authors: Marshall Thomas
Published: May 2005

A methylamine‐incorporating silver stain is described for the detection of deoxyribonucleic acid (DNA), protein and lipopolysaccharide in thick (3 mm) polyacrylamide gradient gels (4–20 % w/v). Its sensitivity for DNA and protein is more than 100 times greater than ethidium bromide and Coomassie Brilliant Blue R‐250, respectively. It detects 0.01 ng DNA, 0.16 ng of human serum albumin and ng quantities of lipopolysaccharide. DNA gives red bands whereas protein and lipopolysaccharide give brown/black bands. The staining procedure is inexpensive, highly reproducible and overcomes the problem of background stain associated with thick gels. It also gives excellent results with thin (1 mm) gels and can be used to stain a number of gels simultaneously. A simplified staining procedure is also discussed.
An electrophoretic investigation of plasma fibronectin by immunofixation after agarose gel electrophoresis
Authors: Castle Stephen L. , Board Philip G.
Published: May 2005

An electrophoretic technique for the rapid screening of fibronectin from plasma and serum is described. This technique was used to screen nearly 3000 human plasma and serum samples from different racial groups as well as plasma samples from dogs, cats, pigs, goats, mice, donkeys, sheep, horses, camels, cattle and Highland ponies for genetic variants. Only one possible genetic variant, from an Indonesian population, was detected. Other potential uses of the electrophoretic technique are discussed.
Placental affinity for pregnancy‐associated murine protein‐1 examined by crossed affino immuno‐electrophoresis
Authors: Hau Jann , Teisner Bøsrge , Svendsen Per
Published: May 2005

Using crossed affino immunoelectrophoresis and tissue extracts added to the first‐dimension gels the affinity between pregnancy‐associated murine protein‐1 (PAMP‐1) and various tissues was examined. Substances binding to PAMP‐1 in the liver and in the placenta were found to affect the electrophoretic migration velocity of PAMP‐1 when added to first‐dimension gels.
Electrophoresis and recovery of active mRNA from composite ultra low/medium gelling temperature agarose gels
Authors: Fourcroy Pierre
Published: May 2005

The electrophoretic separation of RNA species in composite ultra low/medium gelling temperature agarose is described. The resolution obtained was superior to that observed with the standard medium gelling temperature agarose alone. Poly(A)‐containing mRNA was successfully fractionated and recovered in a biologically active form from the separating media.
Changes of the electrophoretic mobility of human monocytes are regulated by lymphocytes
Authors: Bauer Johann , Hannig Kurt
Published: May 2005

The changes of the electrophoretic mobility (EM) of human monocytes during invitro maturation into macrophages were investigated. Incubation of isolated monocytes for longer than 4 days was always accompanied by an increase of the EM of the monocytes. This increase of the EM could only be inhibited when lymphocytes were incubated together with the monocytes. The inhibitory effect of the lymphocytes was optimal when the culture medium was supplemented by autologous plasma and when the cell suspension was kept in teflon beakers, but it was reduced when the culture medium was supplemented by fetal calf serum (FCS) and when the cell suspension was kept in polystyrene dishes. The macrophages, which showed increased EM after 10 days of incubation, had equal antibody‐dependent cell‐mediated cytotoxic (ADCC) activity but lower plaque‐forming activity than the macrophages which retained their original EM during incubation. The finding of macrophage sub‐populations which differ in their EM provides a good opportunity to isolate and further investigate macrophages, which may have different biological activities.
Gel electrofocusing in tubes containing Sephadex
Authors: der Lan Birgit An , Ben‐or Sarah , Allenmark Stig , Sullivan James V. , Fitze Paul , Jackiw Andrew
Published: May 2005

Gel electrofocusing (EF) on Sephadex G‐75, which provides relatively non‐restrictive pore sizes and relatively broad protein concentration distributions (which reduces concentration‐dependent aggregation), was adapted for use in glass tubes. This allows for EF with meticulously controlled low temperatures in conjunction with large sample and gel volumes. Specifically, the technique was developed for EF of steroid receptors, which are highly sensitive to temperature (temperature transformed), and whose aggregation behavior excluded the use of polyacrylamide or agarose gels. The technique uses the temperature‐controlled apparatus designed for quantitative polyacrylamide gel electrophoresis, with three additions. These are a) a 12‐place evacuation device for forming simultaneously 12 cylindrical Sephadex gels (either 6 or 18 mm diameter), b) an electrode arrangement capable of maintaining the same regulated voltage across several gel tubes, and c) a jacket and lid of polyurethane foam to insulate the electrolyte chambers of the EF apparatus.
Dansylated proteins as internal standards in two‐dimensional electrophoresis and Western blot analysis
Authors: Lubit Beverly W.
Published: May 2005

Dansylated proteins have been used in combination as isoelectric point and molecular weight standards and included in gel samples for separation by two‐dimensional polyacrylamide gel electrophoresis. These proteins provide invariant points of reference that facilitate the unequivocal identification of proteins separated by two dimensional polyacrylamide gel electrophoresis without affecting resolution. Moreover, immunoautoradiograms of Western blots can be aligned with their corresponding gel patterns with great accuracy. The logical extension of the established usage of dansylated protein standards to these applications is described.
A novel human kidney‐specific protein detected by two‐dimensional electrophoresis: Isolation, radioimmunoassay, and immunohistochemical localization
Authors: Ziai Mohammed Reza , Jackson Peter , Thompson R. J.
Published: May 2005

High resolution two‐dimensional polyacrylamide gel electrophoresis has been used to detect a soluble protein in extracts of human kidney which was not present in the electrophoretograms of other human tissues. This protein, coded kidney‐acidic protein‐60 (KAP‐60) was purified to homogeneity and was characterized as having a native molecular weight of 85000, a monomer molecular weight of 35 000 and an isoelectric point of 5.6. KAP‐60 was devoid of detectable carbohydrate or phosphate groups. Injection of the protein into rabbits produced antisera which were used to develop a radioimmunoassay for the protein and to localize the protein to specific cells within the kidney by the immunoperoxidase technique. A survey of human tissues by radioimmunoassay showed that KAP‐60 was present in human kidney at approximately 150 times the concentration found in human liver and 300 times the level found in other human organs examined. Immunohistochemistry localized the protein to the proximal and collecting tubules in the kidney with no staining of glomeruli or distal tubules. The present study illustrates the use of high‐resolution two‐dimensional electrophoretic techniques in the detection of human tissue‐specific proteins which may prove of clinical value in the biochemical diagnosis of tissue‐specific damage.
Characterization and visualization of the low density lipoprotein (LDL) receptor by ligand blotting using anti‐LDL‐enzyme linked immunosorbent assay
Authors: Dresel Hans A. , Schettler Gotthard
Published: May 2005

This paper describes the visualization of the low density lipoprotein (LDL) receptor by ligand blotting technique using an anti‐LDL‐enzyme linked immunosorbent assay (ELISA). Solubilized membrance proteins from bovine adrenal cortex are subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, transferred to nitrocellulose paper and incubated with LDL. The combination of peroxidase‐con‐jugated second antibody and 4‐chloro‐naphthol/H2O2 allows rapid development of colored bands where LDL was bound to the receptors. Saturation of the LDL binding to the LDL receptor can be demonstrated. This highly sensitive and rapid ELISA avoids the need for continuous supplies of iodinated antibodies.
Non‐linear pH courses with immobilized pH gradients
Authors: Gianazza Elisabetta , Giacon Patrizia , Sahlin Bengt , Righetti Pier Giorgio
Published: May 2005

Examples of increased resolution obtained with non‐linear pH gradients are presented. Discussed in detail is the “ideal” pH course in the range 4–10, based on the statistical distribution of isoelectric points of protein from data in the literature.
Cell electrophoresis: Automatic measurements by laser light scattering with Lazypher
Authors: Steiner Rudolf , Ottmann Oliver , Kaufmann Raimund , Ann Light P. , Hoffmann Walter
Published: May 2005

Automated cell electrophoretic measurements with Lazypher are based on the laser Doppler technique. Experiments are performed with high precision (of 1.2% S. D.) and in short time intervals of 1–3 min. Application to electrophoretic mobility tests with tanned sheep erythrocytes incubated in supernatants from sensitized murine thymus and spleen cells which had been prepared from cultures containing 5 μg purified protein derivative (PPD)/ml caused a reduction of the electrophoretic mobility (EM) of 5% and 13%, respectively. Control spleen cell supernatants caused a 4% decrease in mobility and no effect was observed with supernatants from control thymus cells. Peripheral blood lymphocytes from healthy adults (n = 56) and children with atopic diseases and congenital heart disease (n = 12) are separated into a high mobility peak (μ = 1.24 μmcm/Vs for adults, μ = 1.19 μmcm/Vs for the child group) corresponding to T‐cells and a low mobility peak, μ = 0.93 μcm/Vs, representing B‐cells. The mobility difference between the adult and child T‐cells is highly significant (99%). Electrophoretic measurements of myeloid cells from healthy donors and patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) show that progressing differentiation within this cell line is accompanied by a decreasing mobility. Blast cells from a patient with acute lymphoblastic leukemia (c‐ALL) had an EM, μ = 1.09 μmcm/Vs, distinct from that of myeloblasts in AML (μ = 1.16 and 1.25 μmcm/Vs) and CML (μ = 1.25 and 1.23 μmcm/Vs) and from normal T‐and B‐lymphocytes.
Some more formulations for immobilized pH gradients
Authors: Gianazza Elisabetta , Astrua‐Testori Silvia , Righetti Pier Giorgio
Published: May 2005

Formulations are presented for 4 to 6 pH unit wide immobilized pH gradients requireing the use of only six available Immobiline buffers, without strong acidic and basic titrants. Recipes are also given for the pH 3–10 range.
Computer automated lanes detection and profiles evaluation of one‐dimensional gel electrophoretic autoradiograms
Authors: Pun Thierry , Trus Benes , Grossman Nili , Leive Loretta , Eden Murray
Published: May 2005

A new fast, precise and reproducible method for lanes detection and profiles evaluation of one‐dimensional gel electrophoretic autoradiograms is presented. It is fully automated, and its principles are based on image and signal processing techniques. The operator usually has only to specify the number of lanes that are present in the preparation, but still may retain overall control. In the first phase, the program automatically locates the lateral position of the lanes, and approximates their left and right boundaries by polynomials (which allow curved as well as straight lanes). The operator can then intervene, if necessary, to adjust these positions. In the second phase, the background variations are corrected; two types of density profiles are computed, representing the average density as well as the total amount of material in the lanes. This calculation is carried out with correction for tilt or curvature for each lane. Finally, prinouts are produced that contain graphs of the profiles together with quantitative measurements. Use of this method is illustrated by analysis of electrophoretic gels containing proteins and lipopolysaccharides of varying mobilities. Experiments made on these gels as well as others yield accurate quantitation of the relative amount of each component.
A simple method for drying of sodium dodecyl sulfate‐polyacrylamide disc gels
Authors: Aono Sachiko , Sato Hiroshi , Semba Reiji , Kashiwamata Shigeo
Published: May 2005

A simple method for the drying, on filter paper, of sodium dodecyl sulfate polyacrylamide disc gels is described. This method has been successfully applied to disc gels with polyacrylamide concentrations of 5, 10, and 15%, and also with a linear gradient of 5–15%. The gels dehydrated without any longitudinal shrinkage are suitable for longtime storage and may be used for molecular weight estimation of proteins.
Effect of conductivity and concentration on the sample stream in the transverse axis of a continuous flow electrophoresis chamber
Authors: Miller Teresa Y. , Williams George O. , Snyder Robert S.
Published: May 2005

The resolution of continuous flow electrophoresis systems is generally measured by the spread of the sample bands in the direction of the electrophoretic migration. This spreading assumes an electric field that extends uniformly between the two electrodes. This paper evaluates the cross‐section of the sample bands in the plane perpendicular to the flow and shows that the spread in the direction perpendicular to the migration increased significantly with the applied electic field. Concentrated samples of monodisperse latex particles and vinyltoluene T‐butylstyrene particles in sample buffers of different electrical conductivities were used to map the shape of the sample bands relative to the zero electric field case. As the electric field was applied, the sample band spread from an initial diameter of only one‐third the chamber thickness until it approached the chamber walls where electroosmosis significantly reduced the resolution of separation. It can be shown, however, that it is possible to minimize these distortions by careful sample preparation and experiment design.
Geometrical and electrokinetical radii of an ion
Authors: Michov Budin M.
Published: May 2005

The existence of a geometrical and an electrokinetical radius of an ion is suggested. An equation is worked out representing the electrokinetical radius as a function of the geometrical radius and the thickness of the ionic atmosphere.
Differences in two‐dimensional patterns of cellular proteins from murine T‐ and B‐lymphocytes after mitogenic stimulation
Authors: Braun Andreas , Waldinger Dorothea , Cleve Hartwig
Published: May 2005

Splenocytes from inbred mice strains were stimulated with concanavalin A (Con A) or lipopolysaccharide (LPS) to form T‐ or B‐blasts. Following three days of mitogenic stimulation, immunofluorescence of the splenocytes with a rabbit antimouse‐immunoglobulin antiserum conjugated with fluorescein isothiocyanate revealed an extensive enrichment of T‐blasts by Con A and of B‐blasts by LPS. The cell preparations, however, represented enriched but not pure populations of T‐ or B‐blasts. Two‐dimensional electrophoretic resolution of cellular proteins from Con A or LPS‐stimulated murine splenocytes and subsequent silver staining of the polyacrylamide gels showed approximately 450 spots (polypeptides). Comparative analysis of the resulting patterns revealed 13 differences specific to the T‐cells and 8 specific to the B‐cells. In order to ascertain the reproducibility of the differences of two‐dimensional protein patterns from Con A and LPS stimulated splenocytes, the experiment was performed several times using two different inbred mice strains (C57B1/6 and BALB/c). Interstrain specific differences were also found at a molecular mass of about 68 kDa and at a pI range between 6.1 and 5.85.
Genetic polymorphism of human serum transferrin in the swiss population: Evidence for three “new” transferrin‐variants
Authors: Scherz Rudolf , Reber Beatrice , Pflugshaupt Rolf , Bütler René
Published: May 2005

Genetic polymorphism of transferrin (Tf) was studied in 473 healthy Swiss blood donors and 236 nonrelated individuals engaged in cases of disputed paternity. Gene frequencies were: TfC1 = 0.7727, TfC2 = 0.1634, TfC3 = 0.0573. Several rare alleles were observed, the frequency of the total of which was 0.0066. Three obviously new Tf alleles were detected. The phenotype distribution was in agreement with the Hardy Weinberg equilibrium. Among 113 mother/child pairs no phenotypes contradictory to the mendelian law were observed. The transferrin system represents a useful parameter in routine paternity testing. Its average chance of exclusion (without rare alleles) is 18.19%.
Quantitative reproducibility of measurements from Coomassie Blue‐stained two‐dimensional gels: Analysis of mouse liver protein patterns and a comparison of BALB/c and C57 strains
Authors: Leigh Anderson N. , Nance Sharron L. , Tollaksen Sandra L. , Giere Frederic A. , Anderson Norman G.
Published: May 2005

Using the ISO‐DALT system for two‐dimensional (2‐D) electrophoresis and the TYCHO system for computer analysis of the resulting protein maps, we obtained high quality quantitative protein abundance data from Coomassie Brilliant Bluestained gels of mouse liver samples. High resolution gels allow more than 100 proteins to be measured with coefficients of variation less than 15 %. A comparison of results from two mouse strains (C57BL/6 and BALB/c) and the cross between them (BCF1) shows that a large number of qutive polymorphisms can be detected, and that, as expected, the amount of protein produced in the heterozygote is intermediate between the parental values. The system described is shown to be capable of reliably detecting decreases in protein abundance such as those expected to result from radiation‐induced deletion of one copy of a gene. The implications of these results for the study of gene regulation are discussed in relation to applications in genetics, toxicology, and differentiation.
Electrophoretic analysis of the glycan microheterogeneity of orosomucoid in cancer and inflammation
Authors: Hansen John‐Erik S. , Jensen SøRen P. , Nørgaard‐Pedersen Bent , Bøg‐Hansen Thorkild C.
Published: May 2005

Using crossed affinoimmunoelectrophoresis with free concanavalin A the glycandependent microheterogeneity of the immunoregulatory glycoprotein orosomucoid was examined in serum from 30 healthy individuals, 28 patients with benign chronic inflammation and 56 patients with cancer of various types and stages and before and after surgery. We found significant differences in glycan structure between the different patient groups. Thus the patients with the acute phase reaction of surgery differed from patients with chronic inflammation, and patients with testicular cancer differed from patients with colorectal cancer in having a greater proportion of biantennary glycan orosomucoid. Surgery was found to result in a temporary change in the relative proportions of the three microheterogeneity types of orosomucoid, lasting about one week.
New assay procedure for glutathione S‐transferase using analytical capillary isotachophoresis: A procedure for the simultaneous analysis of reduced and oxidized glutathione together with glutathione conjugates of electrophilic compounds
Authors: Holloway Christopher J. , Battersby Rüdiger V.
Published: May 2005

A procedure for the simultaneous analysis of reduced and oxidized glutathione together with glutathione conjugates of electrophilic organic compounds by capillary isotachophoresis is described. This method can be applied to quality control of such synthetic conjugates, which are commonly employed as affinity ligands in the chromatographic purification of glutathione S‐transferases (EC 2.5.1.18). Using this new assay procedure, it has been found that the glutathione conjugate of 1‐chloro‐2,4‐dinitrobenzene is unstable in neutral to alkaline solution, degrading to glutathione and 2,4‐dinitrophenol. Anomalies in the electrophoretic mobilities of a homologous series of alkylglutathione derivatives were observed. A possible explanation is provided on the basis of the formation of micelle‐like aggregates, this tendency increasing with increasing length of the hyrocarbon chain. A useful and widely applicable aspect of this analytical procedure is as a method of monitoring glutathione S‐transferase. The major advantages of this new assay technique can be summarized as follows: (1) the assay does not depend on optical properties of the acceptor, (2) the reaction can be monitored in the presence of unrefined biological material;aliquots can be injected without pretreatment and (3) the spontaneous oxidation of glutathione in the assay mixture can be monitored simultaneously.
Influence of the state of denaturation on the migration of adenovirus type 2 structural proteins in sodium dodecyl sulfate polyacrylamide gels
Authors: Caillet‐Boudin Marie L. , Lemay Pierre
Published: May 2005

The changes in the migration of adenovirus type 2 structural proteins in sodium dodecyl sulfate polyacrylamide gels following various denaturation conditions or periods have been studied. Several factors such as proteolytic cleavages, disulfide bridges, carbamylation or residual charge effects which could lead to altered migration were screened. Resistant high order structures sensitive to the action of urea might cause these variations. A likely relationship between the difference of apparent molecular weight, frictional coefficient and hydrophobicity has been established.
The use of free flow electrophoresis in the purification of recombinant human tissue plasminogen activator expressed in yeast
Authors: Barth Françoise , Gruetter Markus G. , Kessler Reinhard , Manz Hans‐Jörg
Published: May 2005

Free flow electrophoresis was used for the purification of human tissue plasminogen activator (t‐PA) produced by recombinant yeast cells. This method was employed for three principal reasons: (a) yeast t‐PA purified by immunoaffinity chromatography was not pure enough to carry out protein chemical characterization, (b) the amount of yeast t‐PA available for purification was extremely limited, (c) t‐PA has a strong tendency to adsorb to support material; a support‐free separation method therefore seemed to be a reasonable way to further purify the enzyme. Free flow electrophoresis was first performed using solutions containing the soluble proteins of yeast cell extract, and secondly, using t‐PA fractions prepared by immunoaffinity chromatography, Yeast t‐PA purified by immunoaffinity chromatography and then by free flow electrophoresis is more than 80% pure and suitable for characterization experiments such as glycosylation studies or N‐terminal sequence determination.
Analysis of polymeric proteins by two‐dimensional polyacrylamide gel electrophoresis on one slab in the presence and absence of sodium dodecyl sulfate
Authors: Bloemendal Hans , Jansen Kees
Published: May 2005

The method presented was developed specifically to be used for the electrophoretic analysis of polymeric proteins in order to establish the number of aggregates and that of their subunits. Electrophoresis is carried out on a single slab; without sodium dodecyl sulfate (SDS) in the first dimension and with SDS in the second dimension. The method is possible since for one‐dimensional SDS gel electrophoresis, SDS is only required in the cathode vessel.
2‐Pyridinecarboxylate as counter ion in capillary isotachophoresis of low‐mobility bases
Authors: Jokl Vladimír , Pospíchalová Jaroslava , Polášek Miroslav
Published: May 2005

An electrolyte system involving 2‐pyridinecarboxylate (Pic) buffer as the leading and formic acid as the terminating electrolyte was suggested for cationic capillary isotachophoresis of bases with low mobilities. Parameters of the system were studied for concentrations of the leading K+ ion of 1–30 mmol/L and for the Hpic/Kpic ratios of 3:1, 1:1 and 1:3 in the buffer. The effective mobility of the terminating H+ ion ranged between 3 and 30 X 10−9 m2V−1s−1 depending on the composition of the electrolyte system. The applicability of the system (at optimum concentration of K+ 5 mmol/L and the HPic/Kpic ratio 1:1) to the analysis of bases was characterized by a zone existence diagram and demonstrated by the separation of a model mixture containing tetramethylammonium, tetraethylammonium, tris(hydroxymethyl)aminomethane, ephedrine, codeine, nortriptyline, 1,10‐phenanthroline, aminophenazone and 4‐aminoantipyrine.
Long term stability of colors after silver staining
Authors: Hempelmann Ernst , Kaminsky Ronald
Published: May 2005

Red cell membrane proteins were separated in high resolution sodium dodecyl sulfate polyacrylamide gels and analyzed with a polychromatic silver stain. A simple method for the long term preservation of silver‐stained polyacrylamide gels is described.
Ultrathin‐layer sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis and silver staining of urinary proteins
Authors: Schiwara Hans‐Walter , Hebell Thomas , Kirchherr Hartmut , Postel Wilhelm , Weser Johannes , Görg Angelika
Published: May 2005

Ultrathin‐layer sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis in combination with silver staining is a highly sensitive and rapid method for molecular size analysis of urinary proteins. First results are achieved after 4 h (electrophoretic run, 100 min, followed by Coomassie Brilliant Blue staining, 2 h). After additional silver staining, including a recycling step, the results are ready for evaluation within 7 h. Urine samples do not need to be concentrated. Within a single gel, 25–27 samples can be run side by side under identical conditions and thus compared without ambiguity. By coelectrophoresis of pure proteins and immunological analysis 13 urinary proteins could be identified and for 7 of them the detection limits were determined. Glomerular protein patterns in samples of urine with a protein concentration in the normal range are compared with the concentration of albumin and transferrin measured by a sensitive enzyme immunoassay. Typical renal and extrarenal protein patterns are shown.
Free‐flow electrophoresis in solutions of borax and mannitol
Authors: Shukun Sergei A. , Gavryushkin Alexander V. , Brezgunov Vyacheslav N. , Zav'yalov Vladimir P.
Published: May 2005

Free‐flow electrophoresis of proteins in solutions of borax and mannitol was investigated. The borax solution represents an equimolar boric acid‐NaOH buffer with a pH of 9.2. On addition of mannitol the pH of the buffer decreases, with increasing polyol concentration, accompanied by a slight decrease in conductivity. Using a 2.5 mM borax solution, buffers with pH from 9.2 to 4.5 can be obtained. Under the influence of the electric field the pH and conductivities of these buffers change in the near‐membrane regions of the separation chamber. Despite the changes the buffers are suitable for electrophoresis of proteins in a free‐flow apparatus. The separation of bovine hemoglobin as well as a mixture of bovine hemoglobin, human serum albumin and cytochrome C in borax‐mannitol solutions is demonstrated at 80–100 mA constant current and a residence time of only 2.0–2.6 min.
Identity of Treponema pallidum subsp. pallidum polypeptides: Correlation of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results from different laboratories
Authors: Norris Steven J. , Alderete John F. , Axelsen Nils H. , Bailey Mark J. , Baker‐Zander Sharon A. , Baseman Joel B. , Bassford Philip J. , Baughn Robert E. , Cockayne Alan , Hanff Philip A. , Hindersson Peter , Larsen Sandra A. , Lovett Michael A. , Lukehart Sheila A. , Miller James N. , Moskophidis Matthäus A. , Müller Ferdinand , Norgard Michael V. , Penn Charles W. , Stamm Lola V. , van Embden Jan D. , Wicher Konrad
Published: May 2005

As the first step in a cooperative effort to standardize the identification of the polypeptides of Treponema pallidum subsp. pallidum, the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) results obtained in 16 laboratories were compared. Although it was possible to correlate the positions of 16 of the major polypeptide bands, the cross‐identification of many of the polypeptides was ambiguous, particularly in the low molecular weight range. Two‐dimensional electrophoresis provided an improved means of separating and characterizing T. pallidum polypeptides as isolated molecular species. An approach to the unambiguous identification of treponemal polypeptides was outlined which will utilize two‐dimensional electrophoresis in combination with specific properties attributable to individual proteins, including reactivity with monoclonal antibodies or monospecific antisera, biochemical and structural properties, and sequence information. To demonstrate the feasibility of this approach, two‐dimensional electrophoresis in conjunction with immunoperoxidase staining was used to specifically identify three cloned T. pallidum proteins.
Application of two‐dimensional electrophoresis in comparing the effects of progestins and tamoxifen on proteins of the abnormal uterus
Authors: Guest Julian F. , Elder Murdoch G. , White John O.
Published: May 2005

In the androgenized rat, uterine histological abnormalities associated with unopposed oestrogenic stimulation can be partially reversed following progestin therapy. The effects of progestins and tamoxifen on the uterine protein composition of androgenized animals have therefore been investigated in an attempt to identify hormonally sensitive proteins that reflect changes in tissue morphology. Subcellular fractions prepared from uteri of androgenized animals were analysed by one‐ and two‐dimensional gel electrophoresis. In all fractions, protein changes were observed and the identity of some of these proteins has been tentatively assigned following peptide mapping. Evidence is presented of an apparent correlation between potency to antagonize the effects of unopposed oestrogenic stimulation and an increase in the proportion of cytosolic glucose‐6‐phosphate dehydrogenase and the B form of creatine kinase.
Group‐specific component subtyping: A high resolution method employing short run cathodic drift
Authors: Kuo Sze‐Ern
Published: May 2005

A high resolution polyacrylamide gel isoelectric focusing method is described for group‐specific component (Gc) subtyping. With this method all the common Gc subtypes can be identified and the Gc variant 1A1 bands are clearly resolved from the Gc 1F and 1S bands. The run time of 90 min represents a substantial saving in time when compared to many other methods reported in literature.
Anti‐mitochondrial protein antibodies in a serum from a patient with systemic lupus erythematosus: Specificity and comparison with other anti‐mitochondrial antibodies
Authors: Celis Julio E. , Madsen Peder , Andersen Irene , Andersen Paul , Vayssiere Jean‐Luc , Croizat Bernard , Thiessen Hanne , Nielsen Søren
Published: May 2005

Anti‐mitochondrial antibodies present in a serum from a systemic lupus erythematosus patient have been characterized by indirect immunofluorescence of cultured transformed human amnion cells, and by two‐dimensional gel electrophoresis and immunoblotting. The antibodies which stained mostly mitochondria in transformed human amnion cells reacted with four mouse brain cortex mitochondrial proteins. These proteins, which have been termed a, b, c and d, exhibited apparent molecular weights of 66 000, 49 000, 49 000 and 44 000, respectively. Proteins a and c exhibited charge variants. Surprisingly, most of the proteins recognized by this serum also reacted with sera from a patient with liver cirrhosis and idiopathic pulmonary fibrosis.
Observations and implications on the migration of calmodulin in a two‐dimensional gel system
Authors: Heydorn William E. , Creed G. Joseph , Jacobowitz David M.
Published: May 2005

These studies investigated the effect of calcium on the migration of calmodulin in a two‐dimensional (2‐D) gel system. As reported for one‐dimensional gels, calmodulin migrates on 2‐D gels primarily as a 21 kDa protein in the presence of 0.1 mM EDTA, and as a 15 kDa protein in the presence of 0.1 mM CaCl2. However, under basal conditions (i.e., neither EDTA nor calcium added to the bùffer system), calmodulin migrates on a 2‐D gel as a doublet of 20.3 and 18.7 kDa. These results suggest that sufficient residual calcium may be present in one (or more) of the standard laboratory reagents used in running 2‐D gels to produce this unique migration pattern for calmodulin. These results may also have implications for the migration of other proteins on 2‐D gels.
Gel electrophoresis with discontinuous rotation of the gel: An alternative to gel electrophoresis with changing direction of the electrical field
Authors: Serwer Philip
Published: May 2005

When using agarose gel electrophoresis to fractionate linear DNA longer than 40–60 kilobases (kB), improved resolution by length has previously been obtained by periodically and discontinuously changing the direction the electrical field. This change of the field's direction was accomplished by alternately activating two sets of electrodes. However, some of the procedures used produce inhomogeneous electrical fields and with all of these procedures it is comparatively difficult to either change the angle between the two directions of the field or suppress formation of local pH gradients. To overcome these problems, a procedure is presented for changing the direction of the electrical field by rotating the gel. During agarose gel electrophoresis, a commercially available stepping motor and indexer are used to periodically rotate a gel on a circular disk within a conventional, horizontal, submerged gel electrophoresis apparatus. Because of the homogeneous field used, DNA forms comparatively sharp, undistorted bands. The previously achieved improved resolution of 40–166 kB, linear, double‐stranded DNÀ has been achieved here by rotating the gel. Dependence of length resolution on both the angle of rotation and the temperature has been measured.
Tetramethylurea as a protein denaturing agent in gel electrophoresis
Authors: Zazra James J. , Szklaruk Boris
Published: May 2005

We have examined the utility of tetramethylurea as a protein denaturant in electrophoresis using transverse gradient gels. Its physical properties make it easier to work with than urea and it appears to denature bovine serum albumin in a similar way. Tetramethylurea is a potential substitute for urea in some electrophoretic systems.
Alpha 1‐antitrypsin and orosomucoid phenotyping using allo A lectin‐peroxidase for detection
Authors: Umetsu Kazuo , Yuasa Isao , Taketa Kazuhisa , Suzuki Tsuneo
Published: May 2005

A new method of identifying allo A lectin‐binding glycoproteins separated by isoelectric focusing is described. The method is based on the combination of neuraminidase treatment, isoelectric focusing and Durapore filter blotting followed by detection with allo A lectin. The technique is simple and reproducible for the determination of both the alpha 1‐antitrypsin and orosomucoid phenotypes in genetic and forensic investigations.
Evaluation of recombinant human tumor necrosis factor by cell electrophoresis. II. In vivo effect of recombinant human tumor necrosis factor on cell populations and natural killer‐activity in spleen of tumor‐bearing mice
Authors: Rychly Joachim , Knippel Eberhard , Milleck Jürgen , Schütt Wolfgang , Nizze Horst , Kramp Burghard , Klinkmann Horst
Published: May 2005

The effect of recombinant human tumor necrosis factor (rH‐TNF) on the composition and natural killer (NK)‐activity of lymphocytes in the spleen of normal and tumor‐bearing CBA mice was investigated. The characterization of cell composition was based on the analytical separation of splenic subpopulations differing in electrophoretic mobility. In untreated tunor mice, an increase of the electrophoretically intermediate population was found at the final stage of tumor growth. Treatment with TNF depressed this intermediate cell population in the spleen of tumorbearing mice, the electrophoretic distribution of splenocytes approaching the normal pattern. Also in normal mice TNF treatment affected the electrophoretic pattern. Already the first injection of TNF induced an increase of the intermediate cell population in the spleen. Tumor‐bearing mice showed a decreased NK‐activity at the final stage. In normal but not in tumor‐bearing mice repeated injections of TNF seemed to depress the NK‐activity. Cell electrophoresis represents a useful method for evaluating the effect of TNF on the cell populations in the spleen of mice.
A study of protein synthesis in subpopulations of cultured human epidermal keratinocytes using two‐dimensional gel electrophoresis
Authors: Easty David J. , Patel Ketan , Dover Robin , Dunn Michael J. , Evans David J.
Published: May 2005

A procedure for the isolation of protein markers of epidermal differentiation in vitro is described. Human epidermal keratinocytes were cultured and radiolabelled in vitro. Fractionation was performed according to buoyant density (which reflects the degree of terminal differentiation) using Percoll density gradient centrifugation. Subpopulations of keratinocytes were characterised using light and electron microscopy, and proteins fractionated using high resolution two‐dimensional gel electrophoresis. Radio‐labelled proteins were detected using autoradiography and fluorography. Integral membrane proteins were characterised using Triton X‐114 phase shift extraction. Data from this in vitro study were compared to silver stained gels of samples from intact epidermis (in vivo). We report quantitative differences between 14 specific protein moieties expressed in subpopulations of keratinocytes and identify some of these proteins. The differential expression of these protein markers and their possible use in the interpretation of the keratinocyte maturation pathway in cultured cells from patients with skin diseases are discussed.
A comparison of rapid staining techniques for one‐ and two‐dimensional polyacrylamide gels
Authors: Patel Ketan , Easty David , Bailey Richard , Dunn Michael J. , Evans David
Published: May 2005

Two rapid staining procedures using 8‐anilino‐1‐naphthalene sulphonate (ANS) and Nitro Blue Tetrazolium (NBT) were compared with a Coomassie Brilliant Blue R‐250 staining technique. The methods were applied to protein separations using both one‐ (1‐D) and two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The ANS procedure worked well for both 1‐D and 2‐D gels. The NBT technique, although more rapid, gave satisfactory results only for 2‐D gels. The ANS and NBT staining methods were both compatible with Western blotting so that they could be used for visualisation of the separation profile prior to blot transfer to nitrocellulose.
Electrophoretic and kinetic characterization of two phenotypes of soluble cytoplasmic L‐aspartate:2‐oxoglutarate aminotransferase in human liver tissues
Authors: Kanemitsu Fusae
Published: May 2005

Two phenotypes of soluble cytoplasmic L‐aspartate:2‐oxoglutarate aminotransferase (AST, EC 2.6.1.1), sAST 1 and sAST 2–1, were partially purified from human liver tissues and characterized by electrophoretic and kinetic analyses. sAST 1 had 2 bands upon electrophoresis on a Cellogel membrane, while sAST 2–1 had 3 bands with an additional anodic one. The frequencies of the two phenotypes of sASTs studied with 1,023 Japanese hemolysates were 98.5% for sAST 1 and 1.5% for sAST 2–1. sAST 1 was electrofocused to 9 bands with pI's ranging from pH 5.5 to 6.1 and sAST 2–1 into 11 bands with pI's ranging from pH 5.3 to 6.1. The molecular weights of the two phenotypes were both estimated to be 95 000. The apparent Km values of the sASTs for L‐aspartate with endogenous pyridoxal 5‐phosphate (PALP) were both 5.2 mM and those with added PALP were 5.9 mM for sAST 1 and 5.8 mM for sAST 2–1. The apparent Km values for 2‐oxoglutarate with endogenous PALP were both 0.26 mM and those with added PALP were 0.41 mM for sAST 1 and 0.37 mM for sAST 2–1. sAST 2–1 was more heat‐stable than sAST 1 upon incubation at 65 °C for up to 80 min, while both preparations were similarly denatured by treating with up to 4 M urea at 28 °C for 90 min. The two sASTs reacted with anti‐human AST‐IgG separated from AST‐IgG complexes in serum.
Fast horizontal electrophoresis. I. Isoelectric focusing and polyacrylamide gel electrophoresis using PhastSystem™
Authors: Olsson Ingmar , Axiö‐Fredriksson Ulla‐Britt , Degerman Margareta , Olsson Birgitta
Published: May 2005

PhastSystem, an integrated system for horizontal electrophoresis and isoelectric focusing in small gels, including automated staining and destaining, is described. Buffers for electrophoresis are supplied to the gel from buffer strips made of agarose. The separation bed is cooled by Peltier elements. All conditions of significance to the results, both during separation and development, are controlled by a microprocessor. For separation, nine programs are available, each with 9 steps; for developement, there are nine programs with 20 steps each. In this paper, we also report results using PhastSystem for purity checking and characterization of monoclonal antibodies after affinity chromatography and also for determining their digestion rate with papain.
Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels
Authors: Heukeshoven Jochen , Dernick Rudolf
Published: May 2005

A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05–0.1 ng protein per band. Total staining time with the new procedure is 75 min.
Horizontal two‐dimensional electrophoresis with immobilized pH gradients using PhastSystem
Authors: Görg Angelika , Postel Wilhelm , Günther Siegfried , Friedrich Claudia
Published: May 2005

Protocols for horizontal two‐dimensional electrophoresis with immobilized pH gradients in the first dimension were modified for horizontal micro two‐dimensional electrophoresis using PhastSystem. Different equilibration conditions of the first‐dimensional immobilized pH gradient gel strip prior to second‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were evaluated. Silver stained two‐dimensional patterns were obtained within 3.5 h.
A continuous acetie acid system for polyacrylamide gel electrophoresis of gliadins and other prolamines
Authors: Clements Robert L.
Published: May 2005

A polyacrylamide gel electrophoresis system buffered by acetic acid alone was developed for electrophoresis of prolamines. When applied to gliadin electrophoresis, the acetic acid system produces more bands than does a conventional aluminum lactate‐lactic acid system (using 12 % acrylamide gels). The acetic acid system is relatively simple, requiring a single buffer component that is universally available in high purity.
Properties of thin‐rod immobilized pH gradients
Authors: Gianazza Elisabetta , Tedesco Giuliano , Cattò Nadia , Bontempi Licia , Righetti Pier Giorgio
Published: May 2005

Immobilized pH gradient gel rods, 1.5 mm in diameter, were cast with a manifold connected to high‐precision burettes. The reproducibility of gel length was ca. 1.7 mm. The average standard deviation σx for spot position was 2 mm after one‐dimensional and 5.8 mm after two‐dimensional runs. In order to bring to completion the elution of the salt fronts into the electrode compartments, carrier ampholytes had to be included in the gel formulation at concentrations of at least 0.5–1%, depending on the pH range. The presence of carrier ampholytes, however, was troublesome in two respects: the gel tended to shrink and the cathodic bands drifted with time. Ionic components in the sample were tolerated up to the following concentrations: NaCl 8 μmoles, sodium dodecyl sulfate 10 μg per tube. In presence of non‐ionic detergents, the gels moved as a whole towards the cathode.
Orosomucoid 1 and orosomucoid 2 types in the Taiwanese and Japanese: Evidence for five new orosomucoid variants
Authors: Umetsu Kazuo , Yuasa Isao , Chen Eng‐Rin , Kudo Toshiyuki , Suzuki Tuneo
Published: May 2005

The genetic polymorphisms of orosomucoid, ORM1 and ORM2, were analyzed by isoelectric focusing on polyacrylamide gels and subsequent immunoprinting. Sera from 600 unrelated individuals in Taiwan and Japan were examined. Five new alleles, designated ORM1*7, ORM1*8, ORM1*4.2, ORM2*8 and ORM2*10, were observed together with common and rare alleles that have been found in the Japanese and the Filipinos. As compared with the Japanese, the Taiwanese have a higher frequency of ORM1*1, and a significantly lower frequency of ORM1*2·1.
Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250
Authors: Neuhoff Volker , Arold Norbert , Taube Dieter , Ehrhardt Wolfgang
Published: May 2005

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G‐250 and R‐250. The new method is based on addition of 20 % v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described [1]. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate‐electrophoresis and isoelectric focusing in carrier ampholyte‐generated pH gradients.