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  • 1
    Electronic Resource
    Electronic Resource
    Improved rehydration procedure for polyacrylamide gels in presence of urea: Demonstration of inherited human prealbumin variants by isoelectric focusing in an immobilized pH gradient (1984)
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 379-381 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Polyacrylamide gels containing immobilized pH gradients (IPGs) are usually extensively washed in deionized water after polymerization and dried when longer storage is required. The original volume of the gel is readjusted by rehydration in water or a solution of appropriate composition and subsequent evaporation, if necessary, by using a fan for weight control. A more convenient procedure is to put the dried gels back into their casting mold used for polymerization and to fill the mold with the appropriate rehydrating solution. Rehydration to the original volume is completed within 30 min for gels of a thickness of 0.5 mm. With gels to be rehydrated in the presence of urea, it was found that water enters the gel matrix faster than urea; consequently, adjustment to the original gel volume at the required urea concentration is not possible in an excess of rehydration solution, but is easily achieved by the modified rehydration procedure. It was also possible to infuse a urea gradient into the gel matrix perpendicular to the pH gradient axis. The effect of urea on the patterns of inherited human prealbumin variants is demonstrated.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150050613
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  • 2
    Electronic Resource
    Electronic Resource
    Hybrid isoelectric focusing: Adsorption of proteins onto immobilized pH gradient matrices and desorption by carrier ampholytes (1987)
    Weinheim : Wiley-Blackwell
    Electrophoresis 8 (1987), S. 52-62 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Comparing the separation patterns achieved by hybrid isoelectric focusing in immobilized pH gradients of 1 and 3 meqv/pH/L buffering power, it is demonstrated that a significant proportion of sample protein is lost in gels with the higher buffering power. A buffering power of 1 meqv/pH/L was found to be the lowest acceptable limit for flat bed gel hybrid isoelectric focusing still ensuring adequate control of the immobilized pH gradient. Protein losses were manifested as diminished overall staining intensity, disappearance of individual zones in dilution series, tailing effects and residual material at the application site, all of the above effects being more pronounced for larger than smaller proteins. Adsorption of proteins to charged binding sites of the immobilized pH gradient offers a plausible explanation for the observed phenomena. The visible effects of adsorption could be reduced by the addition of carrier ampholytes, but not by urea or detergents like Triton X-100, 3-(3-chol-amidopropyl)-dimethylammonio-l-propanesulfonate (CHAPS) or octyl β-thio-glucoside, indicating ionic interactions between the immobilized charged groups, carrier ampholytes and proteins. The adsorption phenomena may strongly affect the quantification of proteins in immobilized pH gradients.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150080111
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Horizontal gradient polyacrylamide gel electrophoresis (1981)
    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 49-54 
    Details
    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.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150020108
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  • 4
    Electronic Resource
    Electronic Resource
    Demonstation of human prealbumin by double one-dimensional slab gel electrophoresis (1981)
    Weinheim : Wiley-Blackwell
    Electrophoresis 2 (1981), S. 148-155 
    Details
    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.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150020305
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  • 5
    Electronic Resource
    Electronic Resource
    Avoiding liquid exudation on the surface of rehydrated gels used for hybrid isoelectric focusing in carrier ampholyte supplemented immobilized pH gradients (1986)
    Weinheim : Wiley-Blackwell
    Electrophoresis 7 (1986), S. 251-259 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Recently, we described hybrid isoelectric focusing in rehydrated polyacrylamide gels as an analytical procedure which is based on separation in immobilized pH gradients supplemented with low concentrations of carrier ampholytes (Altland and Rossmann, Electrophoresis 1985, 6, 314-325). When using this procedure at various pH ranges, in the presence of reducing agents and carrier ampholytes at different concentrations and from different suppliers, over prolonged time intervals for separation and at various acrylamide concentrations in the gel, liquid exudation has been observed on the gel surface which may result in a confluent liquid layer adversely affecting the final separation pattern. The effect is demonstrated and procedures are described to overcome the phenomenon by adding polyols like glycerol, sorbitol, sucrose or dextrans to the rehydration solution. Comparable effects are achieved when these components are added at the same concentration by weight rather than by moles. The addition of 10 g % Dextran 8 or 20 g % sucrose or sorbitol to the rehydration solution prevents liquid exudation for several hours. These additives are compatible with high concentrations of urea, with reducing agents and with neutral detergent (Triton X-100).
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150070603
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    Paper (German National Licenses)
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  • 6
    Electronic Resource
    Electronic Resource
    Flattening and/or expanding of pH gradients in isoelectric focusing gels exemplified with PhastSystem (1988)
    Weinheim : Wiley-Blackwell
    Electrophoresis 9 (1988), S. 262-267 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple method of flattening and/or expanding of pH gradients in isoelectric focusing is described for any pH interval desired: to modify pH gradients near one electrode a paper strip soaked with carrier ampholytes is applied onto the gel close to the opposite electrode. In order to flatten central parts of pH intervals paper strips are applied onto the gel at both electrodes. Conditions and criteria (e.g. amount and pH intervals of carrier ampholytes, width and localization of the paper strip, separation period) for optimization are presented with PhastSystem using ready-made gels with three different pH intervals and pI marker proteins (Pharmacia). Examples utilizing erythrocyte lysates are presented.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150090604
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  • 7
    Electronic Resource
    Electronic Resource
    Experimental evidence for flexible slope of immobilized pH gradients poured under computer control (1985)
    Weinheim : Wiley-Blackwell
    Electrophoresis 6 (1985), S. 140-142 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Recently, Altland and Altland (Clin. Chem. 1984, 30, 2098-2103) described three alternative devices for pouring immobilized pH gradients (IPGs) based on simultaneous delivery of a density gradient and a non-density gradient by using high precision burettes under computer control. One of the expected improvements was an unlimited flexibility for the slope of the non-density gradient (e. g. the pH gradient) which is stabilized by the density gradient. The human plasma proteins prealbumin and apolipoprotein AI and some of their inherited variants are demonstrated by double one-dimensional electrophoresis in the sequence polyacrylamide gel electrophoresis followed by isoelectric focusing (IEF) in IPGs of varying slopes in the range of pH 4 to 7. It is shown that the patterns obtained follow the expectations from the gradients drawn on the computer. We conclude that the new devices for pouring IPGs can provide a valuable tool for the easy handling of complex separation problems to be solved by IEF.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150060310
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