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  • 1
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Phycoerythrin, ferritin, urease, β-galactosidase and thyroglobulin, with molecular masses in excess of 200 kDa, adsorb and consequently fail to migrate to, and focus at, their pI positions in electrofocusing in immobilized pH gradients at a total Immobiline concentration of 20 mM while they do focus normally in pH gradients formed by carrier ampholytes. The addition of carrier ampholytes (pH range 3.5-9.5) at concentrations of 0.1 to 5 % to the Immobiline-containing gels reduces adsorption (desorbs) some but not all of the 5 proteins at specific Immobiline concentrations. The adsorption is not due to water redistribution and consequent reduction in gel porosity; nor is it due to conductivity minima across the pH gradient. The hypothesis that the presence of oligomeric Immobiline contributed to the protein adsorption is the subject of the accompanying report.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Five proteins with molecular mass in excess of 200 kDa were found to adsorb onto gels during isoelectric focusing on immobilized pH gradients (IPGEF). To probe for the mechanism of that adsorption, the homogeneity of the six Immobiline preparations used to make IPGEF gels was tested. Five of these Immobiline preparations appear homogeneous in gel filtration of Sephadex G-10. The sixth Immobiline (pK 9.3) exhibits a minor component eluting ahead of the major peak and comprising less than 4 % of the total Immobiline absorbing at 226 nm. The proportion of the minor component increases with column load. Major and minor components when isolated appear to equilibrate with one another. Judging by the results of mass spectrometry, all 6 preparations are free of small aggregates of less than 500-600 Da molecular mass. Ultrafiltration of the Immobiline preparations through a membrane with 500 Da nominal cutoff leads to partial desorption of only 3 of the 5 adsorbed proteins. CHAPS is ineffectual in desorbing the 5 proteins from the IPG gel made with ultrafiltered Immobilines. None of the 6 Immobiline preparations used precipitates ferritin. All large proteins that adsorb onto IPGEF gels in the pH range 4-9.5 also adsorb onto commercial IPGEF gels in the pH range 4-7.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Immobilized pH gradient (IPG) slab gels are conventionally formed by use of two-chamber gradient mixers on pH-neutralized Immobiline mixtures, co-polymerization of the gradient of Immobilines with acrylamide at 50 °C, washing and drying of the gel to its original weight followed by pre-electrophoresis before applying the sample. This tedious procedure was replaced by one using gradient formation by pump, eliminating pH neutralization of the monomer mixture, substituting polymerization at 50 °C for one at the temperature of electrophoresis (10 °C in this study) and omitting washing and drying of the gel prior to use. Carrier ampholyte (CA) containing IPG gels were formed in the same way except that CAs were added to the polymerization mixture. Pre-electrophoresis of IPG gels, formed by either the conventional or the simplified procedure, was found to be detrimental to the IPG patterns of proteins. The simplified procedure also allowed one to conduct IPG electrophoresis in gel tubes, eliminating lateral zone diffusion.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Sequential moving boundary electrophoresis with protons and hydroxyl ions as the sole counterions, consisting solely of six acids (system A) and of six bases (system B), gives rise to natural steady-state pH gradients similar to those predicted by theory (pH 2.4-5.5 for system A, pH 10.3-12.2 for system B). Mixtures of the constituents in system A and system B gave rise to a natural pH gradient spanning the pH range between the terminal electrolytes (pH 2.4 and 12.2) predicted for systems A and B. The predicted boundary displacement is negligibly small. Experimentally the boundary displacement could not be determined in view of the difficulty of separating it from the initial transient state in which the pH gradient is formed, and a final state in which the pH gradient decays.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 8 (1987), S. 286-293 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Three plant viruses: turnip crinkle (TCV), hibiscus chlorotic ringspot (HCRSV) and pelargonium flowerbreak (PFBV), and polystyrene size standards with radii of 22.4-59.4 nm can be stacked within trailing and leading ion net mobilities of 0.059 to 0.273 (relative to Na+). Stacking was carried out at pH 6.50, 0.03 M ionic strength, 50 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, at a gel concentration of 0.4 %, in agarose gel electrophoresis conducted at 1.2 mA/cm2 of gel. Unstacking occurs under the same conditions at gel concentrations ranging from 0.5 to 1.1 % agarose, while it can be brought about between 0.1 and 0.7 % agarose when the pH is raised to 7.27, corresponding to a front moving boundary with a trailing ion net mobility of 0.216 (relative to Na+). Ferguson plots of viruses and polystyrene particles in the discontinuous buffer system are curvilinear and comparable to those obtained in a continuous buffer at pH 6.50 of the same composition and operative pH as that of the resolving phase of the discontinuous buffer. Particle radii and net charge values can be obtained from the non-linear Ferguson plot in the discontinuous buffer system by previously reported methods of computer simulation, but this Ferguson plot presents a more limited data base than that in the continuous buffer since it excludes gel concentrations which yield relative mobility (Rf) values of 1.0. Since computer simulation provides the range of gel concentrations in which both the fiber radius and length [1], as well as the size of the particle [2], remain constant for a particular preparation of agarose, a simplified alternative method of evaluating particle sizes exists. Within that specific gel concentration range, the linear segment of the Ferguson plot can be used to compute particle sizes by an operationally convenient, albeit approximative, method, using the same PAGE-PACK programs of D. Rodbard which are commonly used in the size determination of macromolecules by polyacrylamide gel electrophoresis. The two methods of particle size determination, based on either the entire non-linear Ferguson plot or on its linear segment in the appropriate gel concentration range, yield similar results (average deviation 12 %). The radii of three plant viruses are dependent to different degrees on the presence of Ca++ in the electrophoretic system. Values obtained in the presence of Ca++ are comparable to those found by electron microscopy.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A previous design of an apparatus for extraction and concentration of proteins from gel slices by moving boundary electrophoresis has been simplified, making it into an upper buffer reservoir accessory for the multifunctional gel tube apparatus commonly used for gel electrophoresis. The design of the collection cup has been improved to facilitate assembly and thereby reduce the risk of leakage. The performance of the apparatus was evaluated by the isolation from sodium dodecyl sulfate - polyacrylamide gel electrophoresis of radiolabeled bovine serum albumin and glucagon receptor.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 4 (1983), S. 404-407 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Poly R-480, an amphoteric dye of molecular weight 160 000,is the product of copolymerization reaction and can be expected to yield a polydisperse pattern in polyacrylamide gel isoelectric focusing (PAGIF). Such is indeed obtained in conventional PAGIF using commercial synthetic carrier ampholyte mixtures but not in buffer PAGIF, in which a few discrete dye bands are produced. Upon excision of single bands and segments of zones in buffer PAGIF and PAGIF in SCAMs, respectively, the entire original pattern is reestablished in each case, thereby confirming the artifactual nature of both the simple band pattern as well as the polydisperse patten. The simple pattern in buffer PAGIF prevails in the presence of 8 M urea and 8 mM CHAPS, while the polydisperse pattern obtained in SCAMs is resolved into multiple bands in the presence of either one or both of these agents. These observations suggest: a) hydrophobic and hydrogen bond dye-dye and/or dye-SCAM interactions produce the artifactual polydisperse pattern in PAGIF in SCAMs, b) electrostatic dye-dye interactions are responsible for the artifactual simple band pattern in buffer EF, and c) dye-SCAM electrostatic interactions produce the artifactual multiple band pattern in the SCAM-urea-CHAPS system. These data clearly underline the need for critical experimentation in the evaluation of PAGIF bands and band numbers.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 10 (1989), S. 254-259 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The representative β-hydroxyethylmorpholinium-chloride-bicinate moving boundary with a trailing ion net mobility relative to Na+ of 0.41, detected by precipitation of chloride with silver nitrate, exhibits a decreasing chloride mobility at increasing polyacrylamide gel concentrations from 3.5 to 45 %T, 5 %CBis. This decrease, largely due to an increase of field strength at constant current, is described by a convex plot of log (mobility) vs. %T (Ferguson plot) and signifies that chloride/bicinate are sieved by the gel. In agarose gels, the same plot of mobility vs. gel concentration is constant below 7 % gel concentration, since in those gels field strength and migration rate remain the same within that gel concentration range. Both in polyacrylamide and in agarose gels the displacement rate of the chloride-bicinate boundary as a function of the time of electrophoresis or distance migrated remains invariant within 15 %. The plot of log(mobility) vs. gel concentration extrapolated to 0 %T is 5.85 and 5.41 (10-5 cm2 s-1V-1) for polyacrylamide and for agarose (SeaKem HGT-P, FMC) gels, respectively. The slightly decreased mobility intercept at 0 %T for agarose is presumably due either to the electroendosmotic properties of agarose HGT-P and/or failure to Sufficiently take into account the flattening of the Ferguson plot in the polyacrylamide concentration range below 3 % in which a transition from a gel to a fluid (sol) medium takes place.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 12 (1991), S. 233-240 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: DNA fragments up to 9 kb in size were stacked and separated by polyacrylamid gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis using a discontinuous buffer system. Polyacrylamide gel at pH 8.9, 2°C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 μg/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72-23130 bp, while agarose gels at pH 8.5, 25°C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 μg/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 × TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 × TBE buffer, with is substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification Rf, the migration distance relative to a sharply defined “buffer front” (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened. (iii) The degree of dilution of the DNA sample becomes unimportant.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Polystyrene sulfate and carboxylate particles (19-189 m radius) were subjected to electrophoresis in glutaraldehyde crosslinked polyvinyl alcohol of molecular weight 25.000 and 650.000 Da at various concentrations. The degree of crosslinking is severely limited by the mechanical properties of the gels that deteriorate beyond a glutaraldehyde concentration which decreases with increasing polyvinyl alcohol chain length. The effective fiber radius of the short-chain and long-chain polymer fiber was 45 ± 25 and 131 ± 47 nm, respectively. Thus, these media do not significantly exceed the apparent fiber thicknes of agarose, are more difficult to prepare - but are well-defined synthetic product rather than natural ones, and have the advantage of carrying no net charge and can therefore be expected to exhibit no electroendosmosis.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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