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J S Fawcett

Publications and source records attributed to J S Fawcett.

At least 19 recordsLinked to original sources
PubMed1992

Transverse agarose pore gradient gel electrophoresis of DNA.

Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a 1-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180).

Coloring Agents↗
PubMed1990

Electrofocusing of acid phosphatases from frog liver, using an immobilized pH gradient.

Isoelectric focusing on carrier ampholyte-containing immobilized pH gradient gels was applied (i) to gels submerged in silicone oil on a Peltier cooled apparatus, (ii) to the separation of the higher molecular weight (HMW, Mr 140,000) and the lower molecular weight (LMW, Mr 38,000) acid phosphatases (AcPases) from frog livers. (i) Electrofocusing was conducted on gels submerged under silicone oil cooled and stirred on a Peltier-thermoregulated horizontal gel support plate. This procedure aimed at a) improving the temperature control of the gel by direct contact of coolant with the gel surface, and thus at being able to focus at the maximal field strength and consequently highest resolution; b) preventing evaporation from the gel and c) excluding atmospheric carbon dioxide. Silicone oil submersion did not abolish water loss from the gel into the electrolyte strips during isoelectric focusing, or a rippled gel surface. Absence of water exudation on the ripples noted previously by Atland [1] was observed. (ii) The electrofocusing of AcPases on immobilized pH gradients yielded patterns which remained stationary as a function of time, by contrast to previous analyses on carrier ampholyte generated pH gradients. The total number of enzymatically active components found in the enzyme preparations from different stages of purification and in the isolated HMW and LMW AcPases was 18. The HMW and LMW AcPases focused in characteristic pH ranges and exhibited qualitative and quantitative pattern differences. Their band patterns add up to that of a crude preparation containing both enzymes. Neither polyacrylamide gel electrophoresis (PAGE) at any nondenaturing pH, nor isoelectric focusing in carrier ampholytes with pattern changes due to the pH gradient drift were able to yield that result.

Acid Phosphatase↗
PubMed1989

Toward a steady-state pore limit electrophoresis dimension for native proteins in two-dimensional polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis in linear pore gradients (4.8 to 48% T, 5% CBis) provides for migration arrest, in a practical sense, after about 5000 Vh for proteins of 290 and 450 kDa, but not for smaller proteins over 20,000 Vh. The arrest is not due to inadequate field strength nor is it caused by water redistribution within pore gradient gels. The possibility is being discussed that exponential pore gradients, and a higher or a lower degree of crosslinking suggested by the literature may be remedies for the present failure to arrest the migration of smaller proteins.

Acrylic Resins↗
PubMed1989

Sieving of ionic constituents across moving boundaries in gel electrophoresis.

The representative beta-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) cm2s-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.

Chlorides↗
PubMed1988

A motor-driven syringe-type gradient maker for forming immobilized pH gradient gels.

A motor driven gradient maker based on the commercial model (Jule Inc., Trumbull, CT) was designed for immobilized pH gradient gels to provide small volumes, rapid stirring and delivery, strict volume and temperature control and air exclusion. The device was constructed and by a convenient procedure yields highly reproducible gradients either in solution or on polyacrylamide gels.

Acrylic Resins↗
PubMed1988

The adsorption of large proteins in electrofocusing on immobilized pH gradients: I. Protein specificity and dependence on Immobiline and carrier ampholyte concentrations.

Phycoerythrin, ferritin, urease, beta-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.

Acrylamides↗
PubMed1988

The adsorption of large proteins in electrofocusing on immobilized pH gradients: II. Dependence on the oligomeric state of Immobiline.

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.

Acrylamides↗
PubMed1977

Chemical differences between thyrotropin isohormones.

Amide determinations have been carried out, with a newly developed g.l.c. method, on thyrotropin hormones prepared by isoelectric focusing. The observed differences in amide content fully account for the differences in the electrochemical properties of the isohormones.

Amides↗