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  • Articles  (62)
  • Biochemistry and Biotechnology  (62)
  • Wiley-Blackwell  (62)
  • 1980-1984  (62)
  • 1981  (62)
  • Chemistry and Pharmacology  (62)
  • 1
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A method of testing batches of ampholytes is presented. By using carbamylated charges standards to co-electrophorese with the protein sample in the first-dimension isoelectric focusing gel, one can monitor, after running and staining the second-dimension sodium dodecyl sulfate (SDS) slabs gel, the continuity of the pH gradient. Charge standards can also be used to check the reproducibility of the pH gradient among batches of ampholytes and to modify the new batch with a small amount of a narrow range ampholyte to assure reproducibility of experiments. Ampholytes for comparison were obtained from three major manufacturers.
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  • 2
    Electronic Resource
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 135-141 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A color development system for staining polypeptides in one- and two-dimensional polyacrylamide gel electrophoresis is described. The basis of the Process involves the complexing of silver with polypeptides reactive centers. The reaction is initiated by placing a polypeptide-containing gel, previously equilibrated with an appropriate concentration of silver nitrate, into a reducing solution that contains sodium hydroxide, sodium borohydride, and formaldehyde. After an appropriate time in the reducing solution, the gel is equilibrated through two changes of an enhancing solution that contains sodium carbonate. The sodium carbonate is necessary for optimal color appear in the polypeptide -silver complexes after several hours in the enhancing solution and are best appreciated while viewing over a fluorescent light box that radiates light at 5000°K. The color of each polypeptide-silver complex is clearly visible above the light background of the stained polyacrylamide gel. Colors of stained polypeptide are blue, green, yellow, and red. Subtle shades of colors also appear and thereby allow easy discrimination of overlapping spots of polypeptides in a two-dimensional gel. To illustrate the method's relative sensitively, a two-dimensional pattern of human fibroblast polypeptides is compared with patterns of a duplicate gel that is stained with Coomassie Blue and developed by autoradiography. The sensitivity of the silver stain process is superior to Coomassie Blue and is comparable to autoradiography after in corporation of conventional levels of 35S- methionine. The utility of the procedure for identifying and characterizing human proteins is illustrated by staining human proteins is illustrated by staining human plasma and platelet polypeptides after two-dimensional gel electrophoresis. The gel electrophoresis color development system consists of steps that are simple, reproducible, and sensitive, and most importantly, which yield colored polypeptide-silver complexes that are reproducible from gel to gel and tissue to tissue.
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  • 3
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 148-155 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A double one-dimensional (D 1-D) electrophoretic method for slab gels with polyacrylamide gel electrophoresis (PAGE) followed by polyacrylamide gel isoelectric focusing (PAGIF) is described for the demonstration of prealbumin (PA) from human sera. Almost pure PA is obtained when serum is submitted to Page i an anionic discontinuous (glycine-chloride) buffer system at alkaline pH. After page the Bromophenol Blue-stained tracking dye boundary in the gel containing PA is cut out and the gel strips are overlayed on an isoelectric focusing gel. A final PAGIF pattern of PA is obtained which is hidden by other serum proteins when one-dimensional PAGIF is performed. PAGIF of PA in a gel containing a gradient from 0 to 8 M urea perpendicular to the pH axis reveals the conversion of at least 7 diffuse bands with isoelectric point (pI) values between 4.2 and 4.7 into three sharp zones with pI values of 5.45 for one minor zone and 5.7 for the two other zones lying very close together. Among 1900 sera from adult pregnant women, 2 were found containing a genetically determined PA variant with a pI of 5.9 for a minor and a pI of 6.35 for a more intense zone in addition to the usual pattern. From the variant PAGE and PAGIF patterns and their distribution in the families of the carriers, it is concluded that PA is a tetramer under control of an autosomal structural gene which remains stable during PAGE abut dissociates into the monomers during PAGIF in the presence or presence of urea it is followed that PA under physiological conditions possibly forms complexes with more components than expected from its known binding capacity for thyroxine and retinal binding protein (RBP). This highly resolving K 1-D electrophoretic technique allows the simultaneous analysis of up to 96 samples under identical experimental conditions. It is concluded that PA may be a good candidate for human mutation monitoring at the protein level.
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  • 4
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 161-168 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple and versatile optical technique for viewing discontinuities in the thickness of horizontal isoelectric focusing gels is described. The discontinuities in the gel are reproducible and relate directly to the ampholyte distribution. Characterization of the capture of the discontinuities in the gel is presented as well as a demonstration of how they may used for localization of focused proteins.
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  • 5
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Alternative technical solutions of the concentration of protein samples or the recovery of proteins from gel slabs by displacement electrophoresis are demonstrated. The samples are collected at a boundary between high-mobility leading ion and a low-mobility terminating ion either in a small Sephadex G-25 columns or in free solution in a rotating-tube free electrophoresis apparatus. The boundary is kept stationary during electrophoresis by a counter flow of leading solution. Spacers and colored markers can be used to facilitate the collection of the proteins. Investigations showed that electroomosis is a very local phenomenon and that, as expected, the magnitude of the effects depends on the conductivity (field strength) of the solution. This implies that electroomosis will vary among different zones in a discontinuous system (as in displacement electrophoresis). Therefore, in most experiments om free solution it is very advantageous to arrange the system such that electrophoresis does not occur.
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  • 6
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 191-191 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 7
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In a study of the biochemical mechanisms of renal toxicity of acetaminophen, quantitative studies of the nonspecific kidney esterases were carried out in New Zealand white female rabbits. After the administration of a sublethal dose of 2.5 g/kg acetaminophen, four animals each plus four controls were sacrificed at 24 and 48 h. subsequent isoelectric focusing of kidney homogenate supernatants over a pH range of 3.5-8.0 showed marked changes in each of the groups. Zymograms of the nonspecific esterases in each group showed a characteristic pattern which deviated greatly from control animals. A morphological and histochemical examination of kidney sections from all animals showed no pathological changes in any of the kidneys. This would indicate that abnormal biochemical changes are being manifested in the kidney long before any morphological deviations. It would seem that this group of enzymes could provide a valuable market for early toxicological changes.
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  • 8
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 174-183 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Ultrathin-layer isoelectric focusing in polyacrylamide gels on silanized supports provided the means for developing a miniature system combining short focusing time with high resolution and operational simplicity. The essence of miniature ultrathin-layer isoelectric focusing is the combined use of short separation distances (1-3 cm) and high field strengths (400-700 V/ cm), which can be applied in standard equipment simply by reducing the thickness of the gel to 20-50 um for improved heat dissipation. By the criterion of coalescence of proteins migrating from different positions, a steady state is reached in pH 4-9 servalyt T carrier ampholytes on 1 cm gels after 2 min (15 Vh) and on 3 cm gels after 10 min total focusing time (110-130 Vh). On 3 cm gels the resolution is 0.03-0.035 pI in wide-range carrier ampholytes, and 0.01-0.02 pI in narrow-range (0.3 pH units) carrier ampholytes isolated by preparative isoelectric focusing of pH 4-9 Servalyt T in Bio-Gel P-60 layers. Miniature ultrathin-layer isoelectric focusing can easily be adapted to the analysis of multiple samples. When the volume is limited to 0.2-0.4 μl, up to four samples per cm can be applied directly on the gel surface. At this loading, the gel volume per focused sample amounts to only 0.5-5 μl. The gel volume per sample is reduced by a factor of 10-200, relative to the 10-12 cm separation distance in 50-100 μm gel layers. This results in a considerable saving of carrier ampholytes and reagents for gel preparation. For serial dilutions of pH marker proteins, the sensitivity of protein detection after staining with Serva Blue G or Serva Violet 49 is 10-15 ng protein in the miniature system. Miniature ultrathin-layer isoelectric focusing should prove to be an attractive method for routine applications in clinical, industrial and research laboratories in those cases when only small amounts of material are available or when rapid results are required. The method is particularly suitable for studies of genetic polymorphisms of proteins and enzymes.
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  • 9
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 10
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 203-212 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Details are given of a method of casting multichannel tubing in silicone rubber in the form of a slab 2 mm thick and up to 380 mm long. Up to 264 parallel channels were formed at 0.8 mm centers along the length of the material, each channel being 0.4 mm in diameter. The material was used both as the outlet manifold in an apparatus for continuous-flow isoelectric focusing (IEF), the construction of which is described, and also in conjunction with a slow-running peristaltic pump as multichannel pump-tubing when it was found that at a flow-rate of about 0.3 ml h-1 per channel, the variation between channels was less than 0.6%. The coupling of the pump-tubing to the outlet manifold of the separation cell of the IEF apparatus resulted in a very smooth laminar flow of liquid in the separation cell itself.
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  • 11
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 213-219 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Polymerization kinetics of polyacrylamide gels, cross-linked with the following: N,N′-methylenebisacrylamide (Bis), N,N′-bisacrylylcystamine (BAC), N,N′- diallyltartardiamide (DATD), N,N′(1,2-dihydroxyethylene) bisacrylamide (DHEBA) and ethylene diacrylate (EDA), have been studied. The gels are polymerized directly in a spectrophotometer quartz cuvette and the kinetics followed at 283 nm (disappearance of the double bond) or at 600 nm (Tyndall effect due to turbidity of Bis, DHEBA and BAC gels). The order of reactivity of the various cross-links appears to be: Bis ≅ DHEBA 〉 EDA ≅ BAC ≫ DATD. The last cross-link, (DATD), was found to actually be an inhibitor of gel polymerization, leading to highly unpolymerized gels, especially at high %C values. Bis and DHEBA gels, at 3 to 5 %C levels, are fully polymerized within 30 min at room temperature, while EDA and BAC gels, in the same %C range, require at least 3h. All cross-links, when used above 10 %C, display quite slow polymerization kinetics, requiring overnight reaction for good conversion of monomers into polymer chains. When polymerizing liquid linear polyacrylamide (without cross-link), an initial concentration of 〉 5% should be used and the reaction continued overnight, at room temperature or higher, for acceptable polymerization efficiency. On the basis of these data, a new structural model for highly cross-linked gels is proposed.
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  • 12
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 1-11 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 13
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Analytical capillary isotachophoresis has been applied as an analytical tool for the assay of the microsomal enzyme UDP-glucuronyltransferase, which is an important system for the detoxification of drugs and many endogenous and exogenous toxins. The substrate donor molecule UDP-glucuronic acid, various products of hydrolytic cleavage, and the products of glucuronidation β-glucuronides, can be assayed in a single run. Furthermore, the glucuronidation of mixtures of acceptors can be monitored simultaneously, since the various β-glucuronides are separated from one another. This method offers a more comprehensive assay of glucuronidation reactions than has been possible using previously published procedures.
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  • 14
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    Electrophoresis 2 (1981), S. 31-39 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: An asymptotic solution to the partial differential equation governing solute behavior in the continuous flow electrophoresis device is combined with solutions for the convective, electrophoretic and electroosmotic velocities in the chamber under nonisothermal operating conditions. The results illustrate the effect of temperature gradients on the net dispersion of solute in continuous flow electrophoresis. Three modes of operation are tested for this chamber: the convective velocity is in the same direction as gravity, the convective velocity is in the direction opposite to gravity and the effect of gravity is negligible.
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  • 15
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    Electrophoresis 2 (1981), S. 12-24 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Membrane proteins and cytosol proteins from organs (liver, brain) of inbred mice were separated by two-dimensional electrophoresis (2DE). The 2DE technique revealed high resolution of complex protein solutions by staining the proteins. The 2DE patterns of membrane proteins showed about 660 protein spots (polypeptides) for both liver and brain. The protein patterns of the cytosols revealed about 800 polypeptides for the liver and about 680 for the brain. The membrane proteins and cytosol proteins of the 2DE patterns represented two protein populations specific for these two cell components. They matched in 7 % (liver) or 23 % (brain) of the total number of membrane protein spots. The matched spots represented dissociable membrane proteins rather than contamination of the isolated membrane fractions by cytosol proteins. Protein patterns of the same cell fraction but from the two different organs investigated contained more than 50 % organ-unspecific polypeptides.Several conditions of our 2DE standard procedure were reinvestigated to test the usefulness for our technique of some methodical steps employed in 2DE by other authors. We found that many steps often used in sample preparations impaired rather than improved the 2DE pattern. In particular, addition of SDS to the protein sample resulted in a considerable loss of proteins during isoelectric foucing. The 2DE itself could be simplified by omitting several steps such as prefocusing, equilibration of focusing gels, forming gel gradients, autoradiography. This has some implications for the development of 2DE to a routine method.
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  • 16
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    Electrophoresis 2 (1981), S. 39-45 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A method for continuous preparative electrophoresis is presented. It is based on the principle that a supporting medium, e.g. a polyacrylamide gel, is moved continuously relative to an electrical field. At one fixed point, the sample is applied to the gel. The charged particles migrate with different velocities in the supporting medium. Simultaneously, they are dragged along with the supporting medium. This procedure leads to a two-dimensional separation of charged particles, which is necessary for a continuous operation of an electrophoresis apparatus. Three different designs of apparatuses, using this method, are presented in this paper. The operation of one construction was tested by separating three protein mixtures.
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  • 17
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    Electrophoresis 2 (1981), S. 45-48 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Mouse splenic lymphocytes were examined for electrophoretic mobility by an automatic cell electrophoresis. The whole population of lymphocytes showed a bimodal histogram of electrophoretic mobility. In contrast, adherent cells to a nylon wool column (B cell-rich) and nonadherent cells treated with anti-mouse IgG and complement (T cell-rich) gave a unimodal histogram. A linear relationship existed between the electrophoretic mobility of mixture of T and B cells and their ratio detected by the method of immunofluorescence, suggesting that the ratio of T to B cells can easily be determined from the electrophoretic mobility of lymphocytes.
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  • 18
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    Electrophoresis 2 (1981), S. 49-54 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple Technique for horizontal gradient polyacrylamide gel electrophoresis (PAGE) in discontinuous buffer systems is described, in which one-dimensional, two-dimensional and double one-dimensional separation patterns can be obtained. The advantages of this technique are: (i) that samples can be applied by overlaying on a variable broad area of the concentrating gel, (ii) that the complete separating gel and the contact with the concentrating gel remain sandwiched between glass plates from the moment of gel polymerization till the end of electrophoretic separation, (iii) that many gels are poured from one gel solution, and (iv) that the gels can be stored, due to an identical buffer composition in the concentrating and separating gel.
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  • 19
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    Electrophoresis 2 (1981), S. 55-59 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Bovine Submaxillary mucin (BSM) and human submandibular mucin (HSM) were electrophoresed in a system in which the mucins were incorporated into an agarose sample gel and separation was carried out in a 5 % polyacrylamide gel. BSM was resolved into two protein- and carbohydrate-containing bands, and a number of non-mucin proteins. When the electrophoresis was carried out at 80°C, the mobilities and resolution of the bands were increased. The entry of HSM into the running gel was also increased at the elevated temperature and the samples were separated into at least four protein- and carbohydrate-stainable bands. Protein bound sialic acid remained unchanged unter these conditions and aggregating activity for cells of a strain of oral streptococcus could be recovered from the gels after the electrophoresis. The facilitation of the electrophoresis of human mucus glycoprotein's at high temperature may reflect the dissociation of macromolecular aggregates of the mucins under these conditions. A number of purified, non-mucin proteins also moved as discrete bands at 80degreeC. It is suggested that electrophoresis at elevated temperatures should be considered generally for the analysis of multimolecular systems that are of biological importance.
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  • 20
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    Electrophoresis 2 (1981), S. 60-63 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple procedure is described for counting labeled protein bands after polyacrylamide slab gel electsrophoresis. Proteins labeled with radioisotopes are separated on conventional polyacrylamide slab gels. After fiction and staining, the slab gels are dried onto filter papers under vacuum. The labeled protein patterns are recorded by autoradiography or fluorography. The sample tracks are separated from each other by cutting the dried slab gel lengthwise. Each sample track is cut into predetermined sections by making parallel cuts with a film cutter. The sections are deposited into scintillation vials and digested with 50% hydrogen peroxide. The digested samples are counted in an aqueous scintillator. This procedure provides a direct quantitation of 3h and 14C-labeled protein bands by reproducibility counting. The results also show that both high resolution and excellent reproducibility are obtainable by this relatively fast and inexpensive method.
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  • 21
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    Electrophoresis 2 (1981), S. 63-63 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 22
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    Electrophoresis 2 (1981) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 23
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    Electrophoresis 2 (1981), S. 65-75 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 24
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    Electrophoresis 2 (1981), S. 76-81 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A new fluorescent staining technique using o-phthaldialdehyde(OPA) has been developed for the rapid post-electrophoretic location for separated zones of proteins and glycoproteins. The method has also been applied to the detection of immunoprecipitates and, in the form of a spray reagent, to the detection of proteins separated on thin-layer gel filtration plates. Major protein zones can be visualized within 1-2 min with maximum sensitivity being achieved within 15 min. In polyacrylamide and agarose gels, the sensitivity is of the same order as staining with Coomassie Blue G-250. Reagents are very inexpensive and, because the reaction is performed at neutral or weekly alkaline pH in aqueous buffers, retention of enzymatic or other biological activity should be optimal. The method is thus particularly suitable for use in preparative applications and when it is desirable to combine general protein detection with specific location of zone of enzyme activity.
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  • 25
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The excellent solublizing properties of high concentrations of formic acid solutions for polypeptides and proteins, including those with high hydrophobicity, are used in polyacrylamide gel electrophoresis. The technical handling of the system is described in detail. The structural polypeptide of poliovirus type1, VP1, VP2, VP3 an dVP4, until now separable in polyacrylamide gel rods containing 70% formic acid. By two-dimensional analysis, namely formic acid electrophoresis in the first dimension and SDS electrophoresis in the second, we have shown that the electrophoretic mobility in formic acid is of the order VP2-VP1-VP3-VP4. with several marker proteins we have also demonstrated that the migration velocity depends not only on the molecular weights but also on the secondary structure of the proteins, especially on disulfide cross-links. High concentrations of salts (3 MCsCI or 6 m Guanidine hydrochloride) in the protein sample do not change the separation pattern, which, however, is strongly affected by 6 m urea.
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  • 26
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple technique for characterization and identification of proteins after high resolution two-dimensional (2D) electrophoresis is described. Poliovirus proteins were separated by 2D analysis and then subjected to llimited proteolysis in sodium dodecyl sulfate (SDS) by alfa-Chymotrypsin or Staphylococcus V8 protease. The cleavage products were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The pattern of peptides obtained was characteristic for a given protein and for the protease used and was highly reproducible. Alfa-Chymotrypsin produced fewer and larger peptides and was suitable for large poliovirus proteins. Staphylococcus V8 protease yielded smaller peptides and therefore showed a higher degree of discrimination between the poliovirus proteins analyzed. A precursor product relationship for a number of poliovirus proteins was determined by this simple technique.
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  • 27
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A zwitterionic methacrylamide derivative, (3-sulfopropyl)dimethy1(3-methacrylamidopropyl) ammonium inner salt (MAPS), was synthesized and then copolymerized with N,N′-methylenebisacrylamide (Bis) to make gels carrying covalently bound sulfobetaine groups. Gels were also prepared with the addition of (3-sulfopropyl)trimethyl ammonium inner salt (TMAPS) to Bis-crosslinked polyacrylamide prior to polymerization. The properties of these gels in gel electrophoresis and electrofocusing were tested and compared with those of Bis-crosslinked polyacrylamide.In MAPS gels the absolute migration velocities of proteins and dyes at either polarity of migration were decreased. Anionic species were much more strongly retarded than cationic species. Ferguson plots (based on protein mobilities relative to dye in a continuous buffer) of catalase at pH 4 obtained on MAPS gels had a similar slope but lower y-intercept compared with those on equivalent polyacrylamide gels, indicating that the available (effective) net charge on the protein was decreased because it interacts with the zwitterionic charge on the gel. Similarly, the addition of TMAPS to polyacrylamide decreased the relative catalase mobility at non-restrictive gel concentrations. However, at gel concentrations above 4 %T, relative mobilities were increased, presumably because the effective pore size of the gel was increased through inhibition of polymerization by the zwitterionic compound.MAPS gels did not exhibit electroendosmosis by the criterion of cyanocobalamin displacement. Resolution between peptides in MAPS gels, and between proteins in ternary copolymer gels made of MAPS, Bis and polyacrylamide, was similar to that on polyacrylamide. Bands of catalase appeared sharpened in MAPS gels. pH Gradients form, decay and exhibit conductance gaps in MAPS electrofocusing gels and in polyacrylamide gels in presence of 0.25-1.5 M TMAPS, in a manner which was qualitatively indistinguishable from polyacrylamide.
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  • 28
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    Electrophoresis 2 (1981), S. 104-112 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Since Desialylated rebonuclease (RNase) from human urine has a highle alkaline isoelectric point (pI 9.5-10.5), an acidic buffer system is required for electrophoretic analysis of different urine specimens. In a common acid disc electrophoresis system in thin-layer polyacrylamide gel, the cathodal mobility of the RNase enzyme(s) was markedly reduced from that expected on the basis of the estimated pI. Dialysis urine against water had a very detrimental effect on the separation of the isozymes, and the addition of buffer to the dialyzed urine further exacerbated this effect. The urin RNase exhibited anomalous electrophoretic behavior in the form different urine specimens or from the same urine specimen adjusted to different concentrations. Treatment of urine with various cations had a significant, positive effect on the migration and resolution of the isozymes. This and other experimental evidence is consistent with the hypothesis that the anomalous behavior was due to the binding of anions from the buffer. A standardized protocol was developed for processing urine specimens, including treatment of urine with myoglobin, which abolished the artifactual variation observed previously and enabled reliable electrophoretic separation of the urine RNase isozymes. In addion, myoglobin treatment had a similar positive effect on the electrophoretic behavior of RNase enzymes from some other sources.
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    Electrophoresis 2 (1981), S. 113-116 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A two-dimensional crossed immunoelectrophoretic method for the determination of the specificity of antibodies raised against rat serum lipoproteins and apolipoproteins (Apo) is described. The method may also be used for the detection of as yet undesignated apoproteins, associated with the serum lipoprotein fractions, and exploits the superior resolving capacity of 7m urea 10% polyacrylamide gel electrophoresis for the first dimension separation and minimizes physical separation of polyacrylamide and agarose in the second dimension electrophoresis by use of low electroendosmotic flow agarose. The use of polyacrylamide instead of cellulose acetate in the first dimension permits the analysis of larger amounts of antigen protein, crossed imunoelectrophoresis of Apo E which binds to cellulose acetate, and the crossed immunoelectrophoresis of antigens previously focused to their isoelectric points in polyacrylamide gels containing ampholytes.
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  • 30
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    Electrophoresis 2 (1981), S. 116-122 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Capillary isotachophoresis has been applied as an analytical monitor during the synthesis of the C-terminal pentapeptide, Gln-His-Phe-Ala-Asn, of bombinin. The samples were separated as cations since the C-terminal carboxyl group was modified to an uncharged amide. Potassium and beeta-alanine were employed as leading and terminating ions and acetate was the common counter-ion. The analyses demonstrated that the stages of synthesis up to the tripeptide, Phe-Ala-Asn, gave quite pure products, whereas a considerable amount of impurity was detectable after the introduction of His into the molecule. From the peptide chemist's point of view, this amino acid presents special problems. However, the impurities found in the isotachopherograms were not detectable by conventional methods such as thin-lyer chromatography. The rapid and sensitive method of isotachophoresis should prove useful to the peptide chemist for assessment of his products, and for redesign or modification of non-optimal synthetic strategies.
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    Electrophoresis 2 (1981) 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 32
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    Electrophoresis 2 (1981) 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 33
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    Electrophoresis 2 (1981), S. 125-126 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 34
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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    Notes: We have designed treatment conditions which allow analysis of the coat proteins of bacteriophage T4D+ by high resolution two-dimensional electrophoresis. The best results were obtained when the mature phage particles were subjected to hypertoniclysis and DNase treatment before solubilization with sodium dodecyl sulfate (SDS). Samples containing 250 ug protein gave the optimum resolution, producing 45 spots as visualized by Coomassie Blue staining. Computerized densitometry measurements showed that these proteins range in relative abundance from less than 0.22% to 11.8% of the total.
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  • 35
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  • 36
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    Electrophoresis 2 (1981), S. 141-147 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Optimal silver staining of proteins in polyacrylamide gels is a fine balance between silver deposition on the proteins and the level of background staining. Reproduciable staining requires empirical standardization of many steps in the procedure. Silver staining in the present form does not stoichiometrically stain proteins, unlike Coomassie Blue. Staining intensity does not bear a linear relationship to protein mass. This is demonstrated by densitometric evaluation of both silver and Coomassie-stained marker proteins fractioned by sodium dodecly1 sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Although silver staining is more sensitive than Coomassie Blue, the increment in sensitivity is both different for individual proteins and dependent on protein concentration. Nevertheless it is an excellent method for one- and two-dimensional (2-D) electrophoresis if only minimal amounts of protein are available and only a qualitative or semi-quantitative evaluation is required.
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  • 37
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    Notes: We have studied the use of bacteriophage T4 coat proteins as a complete set of molecular weight and isoelectric point markers for two-dimensional electrophoresis. We present data which support the feasibility of this approach. The potential utility and advantages of this concept are discussed.
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  • 38
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    Notes: A widened use of isoelectric focusing for preparation of large amounts of pure proteins is highly desirable but is severely hampered, inter alia by the high cost of the carrier ampholytes used for creating the pH gradient. Efforts are therefore being made to replace the carrier ampholytes with simple two-component buffers. To this end, a multimembrane electrolyzer, consisting of 22 compartments with attached cooling loops, has been constructed. The choice of membranes has proved to be one of the main problems. With permeable membranes, even small differences in hydrostatic pressure between the ends of the electrolyzer will cause internal liquid flows that prohibit the formulation of a useful pH gradient. Likewise, flow-tight membranes generally give rise to a large internal flow due to electroosmosis. Among the types of membranes so far examined, polycrylamide membranes form an exception. With these membranes, useful and stable pH gradients have been created. There are drawbacks, however, relating to the low mechanical strength and to the difficulty of producing exactly equal membranes, thereby fulfilling the theoretical demand of constant transference numbers for the ions. The experimental observation that even small disturbances, such as variations of the transference numbers or a minute electroosmotic flow through the electrolyzer, may completely destroy the pH gradient, has been related to the mechanism operating during electrolysis of a simple buffer. While a possible local breakdown of a carrier-ampholyte pH gradient is rapidly repaired by the current, a disturbance occurring in a buffer pH gradient can be repaired only by diffusion, which is a slow process.
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  • 39
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    Electrophoresis 2 (1981), S. 220-228 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The temperature at which polyacrylamide gels are polymerized strongly affects the structure and physical properties of this matrix. The suggested temperature of 0-4 °C has been found to produce highly turbid, highly porous and unelastic gels. At temperature of 25 °C of higher, the gels became progressively transparent, less porous and more elastic. This phenomenon is strongly pronounced in N,N′-methylenebisacrylamide (Bis) gels and progressively decreases for N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) and N,N′-bisacrylylcystamine (BAC) gels. It has been attributed to the formation of hydrogen bonds among cross-linker molecules, which are presumably stabilized by low temperature and progressively broken at higher temperatures. The most extensively H-bonded compounds seem to be Bis molecules, since they can form four H-bonds/molecule, followed by DHEBA (in which inter-molecular H-bonds have to compete with intra-molecular H-bonds) and finally by BAC, which is sparingly H-bonded at 2 °C, and fully uncomplexed at 30°C. It is suggested that polymerization at 0-4°C should be abandoned, since it leads to unhomogeneous and unreproducible pore size matrices. Since H-bonds in Bis molecules are fully disrupted only at 60°C (a temperature unsuitable for gel polymerization, since it produces short chains and unelastic matrices), it is also suggested that the use of Bis should be discontinued in favour of better cross-links, such as DHEBA. In all cases, best polymerization conditions are obtained at 25-30°C. For all three cross-linkers, homogeneous gels are obtained in presence of 8 M urea or in pure formamide as solvent, since both agents fully disrupt H-bonds.
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    Electrophoresis 2 (1981), S. 228-235 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: An ultrasensitive silver stain is described for the detection of proteins in 4-20 % w/v polyacrylamide gradient gels of 3mm thickness, Maximal sensitivity was achieved by substituting methylamine for ammonia in the diamine preparation and adopting a strategy of overstain followed by destain. The prediamine steps were performed at 60°C to conserve time, and the intensity of final background stain was reduced by altering the method of diamine preparation. From our experience, the technique described is more sensitive and reliable than previously published methods when applied to gels of this thickness, In any case, the sensitivity in our hands was at least equal to that of the newly described photochemical technique [7].
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    Electrophoresis 2 (1981), S. 247-250 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: When studied by thin-layer polyacrylamide gel isoelectric focusing, alpha1-an-tichymotrypsin (A1AChy) in human plasma presents a microheterogeneous pattern, consisting of seven bands with isoelectric points (pI) between 4.1 and 4.45. After removal of sialic acids by neuraminidase treatment, four bands are seen with a more alkaline pI (about 1.0 pH unit). Thus, the microheterogeneity of human A1AChy is due only in part to differential sialylation of the isoproteins. Incubation of A1AChy (in excess) with alpha-chymotrypsin results in the formation of a primary complex. In the presence of excess protease, incubation results in a secondary complex with a lower pI. Upon incubation of A1AChy with trypsin, no protease-inhibitor complexes are observed, although there is evidence for partial degradation of the A1AChy molecule.
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    Electrophoresis 2 (1981), S. 240-246 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A method is reported for analyzing the oligomeric state of proteins in a mixture by multidimensional electrophoresis. Water-soluble proteins prepared from human erythrocyte membranes were separated by means of gel filtration followed by electrophoresis in detergent-free disc gels. The major bands in detergent-free disc gels were identified by means of electrophoresis into a second-dimensional slab gel containing sodium dodecyl sulphate. Detergent-free disc gels were also subjected to two-dimensional electrophoresis into detergent-free slab gels consisting of a continuous gradient of polyacrylamide gel. It was shown that many of the water-soluble proteins existed as distinct oligomers. Spectrin aggregates (tetramer and dimmer) accounted for the slowest moving bands in the detergent-free disc gels. A water-soluble protein of the component 3 region appeared to be present as a hexamer, while component 4.1 was present as a tetramer. Components 4.3, 4.9 and 5 appeared to be present in a large number of aggregated states. Components 7 and 8 formed a heteropolymeric protein complex.
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  • 43
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    Notes: The rate of pH gradient development and protein migration towards their isoelectric points(pI) were investigated for thin-layer polyacrylamide gel isoelectric focusing (PAGIF) using Pharmalyte 3-10, Ampholine 3.5-9.5 and Servalyt 2-11 as carrier ampholytes. Using volt-hours(Vh) as the controlling parameter, it was found that both pH gradient development and protein focusing proceeded very similarly with all three carrier ampholytes, Only two significant (but minor) differences between the carrier ampholytes were observed: (1) due to a faster voltage rise with Pharmalyte, the Vh needed for focusing are achieved faster than with the other two carrier ampholytes and (2) focusing of protein with alkaline pI's (〉 pH 8) is slightly slower with Servalyt (both in time and Vh). This is presumably explained by the low field strength at the alkaline end of the pH gradient with Servalyt. Also, it was found that pH gradient development was completely independent of applied voltage when Vh were used as controlling parameter.
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    Electrophoresis 2 (1981), S. 258-259 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 45
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    Electrophoresis 2 (1981) 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 46
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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    Topics: Biology , Chemistry and Pharmacology
    Notes: A method of direct tissue isoelectric focusing (DTIF) in agarose of human skeletal muscle is described. This particular method was developed to utilize the small amounts of tissue obtained by needle muscle biopsies performed for diagnostic purposes. 20 μm thick cryostat sections were adhered to the hydrophilic surfaces of small GelBond rectangles. These were then applied directly to the surface of the gels. A charge-balanced purified agarose was used to make the gels, which contained Triton X-100 to enhance protein solubilization. Stabilization of the pH gradient was attempted by employing a 3% w/v ampholyte which was a blend of 0.85% w/v pH 8-10.4 and 2.15 % w/v pH 3-10 Pharmalyte, by using anolyte regulation with 0.1 M aspartic acid and by focusing the gels under a CO2-extracted nitrogen atmosphere. Equilibrium of proteins stained by Coomassie Brilliant Blue R-250 was apparent as monitored by comigration of sections from both anode and cathode. With the exception of the cathode end, excellent resolution and reproducibility was achieved. Better cathode resolution was noted with non-equilibirium conditions. Preliminary zymograms of lactic dehydrogenase (LDH), using a tetrazolium technique, have shown consistent patterns of multiple isoenzymes, the basic components of which were best seen using non-equlibirium conditions.
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  • 47
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    Notes: Rheoelectrolysis is defined as convection-free electrolysis of an electrolytic solution under simultaneous liquid transport from anolyte to catholyte and vice versa. Equal rate of transfer in both directions is assumed in this paper, and hence there is no liquid flow within the electrolyser. If this process is conducted long enough, a steady state can be expected, characterised by mutually balancing flows of ion constituents within the electrolyser (by electric migration) and in external ducts (by pumps). The differential equation of the steady state is deduced, and it is shown that it is easily solvable if transport numbers and diffusion coefficients are constant throughout the electrolyser. In this case the concentration courses become linear. The conditions for the presence of both buffer components in all parts of the electrolyser. are given. These conditions being satisfied, it is possible to calculate the pH course to be expected throug the eleltolyer. It is found that a pH span of about 2.6 unite can be covered by the rheoelectolysis of an odinary buffer solution. The pH gradient is at a minimum at the centre of the apparatus and increases towards both electrodes. The pH courses is sigmoid in shape and appears to be very useful for isoelectric focusing of proteins and other ampholytes. The method may become important in preparative work on a large scale in order to save the cost of carrier ampholytes in quantities.
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    Notes: Steady-state rheoelectrolysis of a salt of a weak base and a weak acid, with a possible excess of base or acid, is treated theoretically. For an accurate deduction of the pH course through the region where the pure salt prevails, the treatment is based directly on the mass action equations and not on Henderson's equation. However, the conductivity contributions of the water ions H+ and OH- are not taken into account, a limitation that restricts the validity of the theory roughly to the pH region between 4 and 10. The pH span that can be covered by this technique is found to be about 2.5 units bigger than the pK difference between base and acid. An exact expression for the pH gradient is deduced, and it is shown that this gradient has a maximum at the location of the pure salt and minima in the neighbourhood of the two pK values. There is no theroretical obstacle to the use of this technique for isoelectric focusing without carrier ampholytes.
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  • 49
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    Electrophoresis 2 (1981), S. 273-278 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: It was recently observed that bovine serum albumin (BSA), adsorbed onto part of the surface of a glass slide, diffuses along the unoccupied part of that surface (without redissolving in the bulk liquid) at a much greater velocity than the normal diffusion rate of BSA, free solution. In this work a study is made of the electrophoretic transphoretic transport of BSA, adsorbed onto part of the surface of glass slide, along the hitherto unoccupied part of the glass surface. The electrophoretic mobility of adsorbed BSA along the unoccupied glass surface proved to be up to ≅ 2 times faster than in electrophoresis on cellulose acetate strips at the same pH and ionic strength, and up to ≅ 1.6 times faster than in moving boundary electorphoresis. The increase in electrophoretic mobility of adsorbed BSA is most pronounced at the highest density of adsorption. At that density the oblong BSA molecules are closely packed, most probably adsorbed in a tight monolayer, with each molecule positioned perpendicular to the glass surface (although other configurations cannot be excluded). The major driving force enhancing the diffusivity, as well as the electrophoretic mobility of adsorbed BSA, appears to be the high surface pressure of he tightly packed BSA layer. A tentative explanation is offered for the low resistance BSA molecules encounter in moving parallel to the glass surface while remaining adsorbed to the liquid, involving free energies of adhesion of individual protein molecules in various orientations.
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    Electrophoresis 2 (1981), S. 287-290 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The effect of voltage on the rate of desorption of radiolabeled rabbit immunoglobulin G (IgG) from protein A-Sepharose was studied. In the absence of voltage, the rate of desorption was measured using a vast excess of unlabeled IgG to prevent re-adsorption. Electrophoretic desorption was measured using a column equipped with electrodes and with eluent flow occurring in the direction of electrophoresis. The rate of desorption was very sensitive to voltage, and, furthermore, desorption could occur at a rate 25 times faster than in the absence of a voltage. The results are interpreted to indicate that the presence of voltage not only altered the equilibrium of the system but altered the equilibrium or rate constants of the system.
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    Electrophoresis 2 (1981), S. 279-287 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The analysis of immunoglobulin by gel electrophoretic methods is presently burdened with two problems: the incapacity of conventional buffer systems to spread the polyclonal immunoglobulin pattern across the entire separation path length in gel electrophoresis, and the appearance of distinct bands in the conventional electrofocusing patterns, instead of he continuum of zones  -  which would be expected form a polyclonal mixture of “charge isomers” and which is indeed obtained in gel electrophoresis. Polyclonal serum immunoglobulin differ from one another predominantly in net charge (i.e., they are “charge isomers”). The operative pH of gel electrophoresis was therefore optimized. The acidic side of he isoelectric operative pH of 5.32, a discontinuous (multiphase) buffer system was found which was capable of stacking all immunoglobulin in a lyophilized preparation.In order to provide a magnified view immunoglobulin heterogeneity, the immunoglobulin mixture was fractionated into 26 groups of charge isomers using preparative isotachophoresis. When subjected to polyacrylamide gel electrophoresis in resolving gels operative at pH 4.85, 0°C, the isotachophoretic fractions spread between relative mobility values of 0.4 to 0.9 in the electrophoresis pattern, i.e., their sum total covered the entire separation path. Under these conditions, isotachophoretic fractions also revealed immunoglobulin oligomers. The number of oligomeric forms increased from increased from the leading (most basic) to the trailing (most acidic) immunoglobulin components within the stack.In polyacrylamide gel electrofocusing of the isotachophoretic fractions of immunoglobulin, using pI-renge 6-9 ampholine, addition of a 0.01 M mixture of amino acids to the carrier ampholytes, and 20h or more electrofocusing time at 20 V/cm of gel, 0°C, polyclonal immunoglobulin patterns appeared continuous. Electrofocusing patterns of those isotachophoretic fractions of immunoglobulin which contained oilgomeric forms (as revealed by gel electrophoresis ) exhibited one or two major bands against the continuous background of stainable components. Monoclonal immunoglobulin produced distinct major bands under the same comditions.Thus, either polyacrylamide gel electrofocusing in the pI-range 6-9 or polyacrylamide gel electrophoresis in a buffer system operative at pH 4.85 appear promising for the development of a clinically practical and artifact-free electrophoresis analysis for specific oligoclonal immunoglobulin.
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    Electrophoresis 2 (1981), S. 291-295 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The kinetics of photopolymerization, in presence of riboflavin or riboflavin-5′-phosphate (FMN), have been studied either spectrophotometrically, at 283 nm, or chemically, by titrating unreacted double bonds with permanganate. The two sets of data are in good agreement and suggest that at least 8 h of light exposure are needed to ensure 95% conversion of monomers into polymers. Up to now, it was generally accepted that photopolymerization should proceed for 1 h. It is not necessary to prolong light exposure for longer than 8 h, because even a 24 h illuminatin period only improves the conversion from 95% to 96%. Although conditions are described which ensure at least 95% conversion of both riboflavin-and persulfate-catalyzed gels, their visco-elastic properties are widely different, suggesting that the final structure of these two matrices must vary considerably as a function of the catalyst used.
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    Electrophoresis 2 (1981), S. 296-303 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Peroxidase isozymes from two flax genotypes were separated electrophoretically using a range of concentration, regression models were fitted by weighted least squares method, using matrix techniques and appropriate programmes, weight were reciprocals of variances of significance, as a chi square, of the residual sums of square (SS). Log (Rm) Plotted against gel concentration showed significant curvilinearity; Log (P/Hb), where P was an isozyme's migration was linear with concentration. Matrix method allowed a completely generalized approach to the analysis of data of this type. In particular, regression data could be readily combined so as to allow orthogonal comparison between Intercepts and slops in an analohgous fashion to the orthogonal breakdown of row, column and interaction effects in a balance two-way analysis of Weighting effected some improvement in comparison between isozymes; combined analyses of weighted regression produced striking improvement in discrimination relative to simple pairwise Comparisons between isozymes.
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    Electrophoresis 2 (1981), S. 304-307 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Six of the most recent silver staining method for detecting polypeptides on polyacrylamide gels were compared. We found the method of Sammons et al. (Electrophoresis, 1981, 2, 135-141) to be least expensive and most reproducible, For staining gels of 1.5 mm in thickness, it is also the most sensitive method. Other methods may be preferable for staining gels 0.8 mm or less in thickness.
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    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple method for the quantitative determination of glycosaminoglycan concentration and radioactive precursor incorporation into glycosaminoglycans is describe. Tissue glycosaminoglycans are separated by electrophoresis on cellulose acetate sheets. Sheets are then impregnated with diphenyloxazole in toluene and tritium activity quantitated by fluorography. Absolute absorbance measurement of X-ray film fluorograms exposed for 80 h demonstrate a sensitivity of 50 dpm per samle (〈 5 dpm/mm2) and reproducibility of 7% Diphenyloxazole is removed with toluene and the sheets are stained with Alcian Blue. Absolute absorbance determination allow quantity measurement of mass with a sensitivity of 0.05 nmol of disaccharide subunit and a reproducibility of 6%. Beer's law is followed to 3nmol. Example of the utilization of this technique in studying radionuclide incorporation into glycosaminoglycans in these tissues- articuular cartilage, uterine cervix and skin-are presented.
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    Electrophoresis 2 (1981), S. 320-323 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A study of properdin factor B (Bf) in 22 North and Central American populations demonstrated gene clusters delineating Old World Caucasian, New World and Black Ancestoral groups. Result of gene frequencies were comparable to data in the literature of European Caucasian and African Blacks. New World indigeneous population were clearly separated from other racial groups with hybrid population clustering in between the major genetic contributions.
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  • 57
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 315-320 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A new, simple procedure is described for the preparation gel matrix for preparativde isolelectic focusing (IEF). 0.5-1% Agarose of IEF grade is incorporated with 2.5% Sephadex G-200 Superfine (SF) and 3% Pharmalyte, yielding a uniform and rifid m atirx. No gel washing, swelling of degassing is necessary. Optimum gel consistency is quickly and easily obtained since no dehydration of the gel timum gel consistency is quickly and easily obtained since no dehydration of the gel slurry is required. Gel preparation time is reduced to 0.5-1 h and gel handling is simplified. Protein can be easily recovered from the matrix. The pH gradient profile and resolving properties of agarose-Sephadex were compared with those of Spphdex G200 SF and IEF-grade agarose. The profiles of agarose-Sephadex and Sephadex matrixes are smoother and extend further in both directions than those for agarose. Focusing in agarose-Sephadex and Sephadex also results in fewer edge effects. The ability to distinguish closely resolved bands in agarose-Sephadex is superior due to its ability to be directly stained and its avoidance of eletroen-domestic effects associated with focusing in sgarose. Changes in voltages enables us to follow the development, stability, and decay of the pH gradient. Agarose-Sephadex and Sephadex pH gradients developed more smoothly and remained more stable than those for agarose. This new support system combines the good focusing properties of Sephades G-200 SF with the simple handling, rapid and uniform gel formation of agarose, while overcoming many of the drawbacks of using either material alone.
    Additional Material: 4 Tab.
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  • 58
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 323-326 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A comparison of specimens tested in our laboratory for the red cell enzymes sub-system PGM1 by isoelectic focusing and conventional electrophoriesis demonstrated 11 rare allotypes. In addition to the allotypes PGM13, PGM16, PGM16 MAL, 16 KADAR, 17 and 18, we indengified five new variants: 1a5, 1a6, 1a7, 1a8, 1a9. Of the five new variants only 1a6 and 1a9 could be separated from common PGM1 variants on conventional elehoresis. Because of the unique banding patterns for the variants observed, it was found necessary to use both isoelectic focusing and conventional methods for proper identification. Due to the presence for several different momeclatures for naming PGM variants, it is felt that a universal nomenclature should be defined to deal with PGM variants using both methods of analysis.
    Additional Material: 7 Ill.
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  • 59
    Electronic Resource
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 327-330 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Ultrathin-layer isoelectric focusing in 100 um polyacrylamide gels is described for the separation of the isozymes of the PGM1-locus. The PGM1-subgroups are best resolved in pH 5-7 gradients obtained by mixing Ampholine and Servalyt carrier ampholytes in a 2:1 ratio. By using a high field strength (180 V/cm) in the final stage of isoelectric focusing, the isozyme bands are sharper than in conventional thin-layer gels. This facilitates identification of the subgroups. Distortions of the pH gradient are avoided if low initial field strengths (15-20 V/cm) are used. The method is highly reproducible and equally applicable for the analysis of blood samples as well as blood stains.
    Additional Material: 5 Ill.
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  • 60
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 330-332 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Electrochromatographic studies of several metal ions on lanthanum antimonateimpregnated papers have been carried out. Various background electrolytes were used for these studies at different voltage and time intervals. On the basis of the differential mobilities of metal ions, which depend on the ion exchange properties of lanthanum antimonite and the nature of complex formation with the electrolytes, some important binary and ternary separations have been achieved.
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  • 61
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    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 335-337 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 62
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 333-334 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A reproducible isoelectic focusing method has been developed which displays the structural proteins of hair. By staining with the photochemically derived stain, hair protein variability was observed. On a limited number of individuals, five different isoelectric focusing patterns were detected. In addition, polychromatic differences of proteins with similar isoelectric points were found.
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