摘要:

AbstractImprovements of isoelectric focusing (IEF) in agarose are described that result in consistently obtaining broad, linear pH gradients in the range pH 4 to 9.2 at equilibrium. Cathodic drift has been reduced by the use of a blend of commercial carrier ampholytes and by incorporating lysine and arginine in the catholyte as spacers. Resolution has also been enhanced by the use of thin gels, supplemented with sorbitol and glycerol. In addition, a comparison of two agarose preparations for isoelectric focusing was performed using two commercial carrier ampholytes. To minimise background staining, Triton complexes were removed by modification of the fixing and clearing procedures. These modifications to IEF in agarose are being applied to direct tissue isoelectric focusing (DTIF) of normal and diseased human skeletal muscle.

ISSN:0173-0835    

DOI:10.1002/elps.1150030602

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractTo detect bacteriophage‐antibody complexes, specific antiserum has been incubated with bacteriophages and antiserum‐induced aggregation has been detected by agarose gel electrophoresis, using differential sieving of the gel to fractionate aggregates by size. Agarose gel electrophoresis should also be of use in monitoring reaction of other virus‐sized antigens with antib

ISSN:0173-0835    

DOI:10.1002/elps.1150030603

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractPolyacrylamide gel electrophoresis has been adapted to investigate bovine serum albumin (BSA) at sub‐saturation concentration levels of sodium n‐dodecyl sulphate (SDS). Two specific complexes were detected which coexisted between SDS concentrations of 0.4 and 5 mM. The binding isotherm for SDS on BSA was determined under comparable conditions by equilibrium dialysis and used in conjunction with the electrophoretic data to determine the composition of the complexes. The complexes had the following stoichiometry, BSA (SDS)5and BSA (SDS)36, and closely resembled two of the three complexes that have been observed by moving boundary electrophoresis. The experiments indicate that the polyacrylamide gel electrophoresis (PAGE) technique when applied below surfactant saturation could be a useful tool in the study of protein‐surfactant interac

ISSN:0173-0835    

DOI:10.1002/elps.1150030604

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe designs of both gel and elution chambers in preparative electrophoresis apparatuses are key elements for their performance. The present paper describes four types of designs which were developed by the author to alleviate the shortcomings found in the preceding designs. Performance tests, using human hemoglobin (Hb), partially purified haptoglobin 2‐1 type (Hp 2‐1), and bis‐albumin‐containing serum, show that insufficient dissipation of Joule heat and a heterogeneous electric field cause curvature and slant of the sample zones, and that only a chamber of optimal shape and size leads to minimal dilution, giving a good resolution. Moreover, notch, angle, and rim (Fig. 5), designed to support the gel, and tapering of the elution chamber to minimize its volume, are also important ministructures for the performance of the instrument. Unoptimal sets of these structures also cause concave sample bands and result in bad resolution and excessive dilution. These findings emphasize that in scaling up the apparatus, the structure must be designed on the basis of the detailed fundamental experiments from macrostructure to microstructure by using models of actu

ISSN:0173-0835    

DOI:10.1002/elps.1150030605

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractAn indirect fluorescence method for locating colorless protein bands in focused Sephadex gel slabs, demonstrated previously using standard protein solutions [1, 2], has been applied to a complex natural protein mixture: preconcentrated human urine. Protein bands separated by distances of 2 mm or less in the gel slab were accurately located and excised without cross‐contamination. Urinary pigment did not interfere with the localization technic. Microgram quantities of six protein bands were excised from thin gel slabs, and then partially characterized by cellulose acetate electrophoresis. In addition, the bands belonging to two homologues of β2‐microglobulin (B2M I and II) were identified immunologically after systematic excision of the various protein bands in the focused pattern. This one‐step protein isolation procedure was scaled up to the milligram level for the isolation of B2M‐II. In two experiments, 1.60 mg and 2.22 mg were isolated in 73 % and 78 % purity, with yields of approximately 55 %

ISSN:0173-0835    

DOI:10.1002/elps.1150030606

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractRheoelectrolysis of a salt of a weak acid with a weak base with controlled liquid exchange between anolyte and catholyte has been found to give pH courses in grave disagreement with theory. In addition, they were constantly drifting towards lower pH values. These results illustrate experimental difficulties in effectuating conditions compatible with the postulates of the theory: the drifting pH course was due to successive anodic destruction of the base, which gradually disappeared from the system, contrary to one postulate of the theory. The disagreement between the unfavourable experimental and the favourable theoretical pH course was traced to an unintentional internal liquid flow from anode to cathode, in contrast to another postulate of the theory. For that reason, the differential equation for steady‐state rheoelectrolysis with internal liquid flow is deduced and solved. The concentration courses of the ion constituents are found to be exponential instead of linear, which explains the development of an almost constant pH within a great portion of the apparatus and a steep pH change within the remaining portion. Theoretical pH courses for rheoelectrolysis with internal liquid flow are constructed and found to resemble the experimental curves very much. Quantitative information on the maximum tolerable internal flow is give

ISSN:0173-0835    

DOI:10.1002/elps.1150030607

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractContinuous gradient gels in capillaries with acrylamide concentrations ranging from 2–20 % or alternatively from 2–40 % are equally suited for the fractionation of complex mixtures of macromolecules in aqueous solutions within a range of molecular weights from 103‐106. A time‐saving method for the simultaneous preparation of up to 48 identical linear micro‐gradient polyacrylamide gels is described. With this method it was possible to compare the patterns of aqueous extracts and to determine tissue‐specific bands of the mossFunaria hygrometrica L. Sibth.in the low molecular weight range for the

ISSN:0173-0835    

DOI:10.1002/elps.1150030608

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe genetic variability of the human group‐specific component (Gc) system has been examined by isoelectric focusing with immobilized pH gradients. The phenotypes of the six common Gc types and of 15 rare genetic Gc variants are described and presented. High resolution of the Gc components is accomplished. The classification can be made without specific immune reaction. Isoelectric focusing with immobilized pH gradients represents a powerful tool for analyzing genetic variabilit

ISSN:0173-0835    

DOI:10.1002/elps.1150030609

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe McIDAS computer system (Man‐computer Interactive Data Access System) at the University of Wisconsin was used to analyze double‐label autoradiographs of two‐dimensional (2‐D) electrophoresis gels. The 4 samples used to generate the 2‐D gels were obtained from 8 vitamin D‐deficient rat intestines 25 h after injection with 1,25‐dhydroxyvitamin D3(1,25‐(OH)2D3) or vehicle. To perform the analysis, the patterns on Kodak XAR5 (XR) film, which detects14C +3H by fluorography at ‐75 °C, and on Kodak NS‐2T (NS) film, which detects14C only by direct autoradiography at room temperature, were converted to digital images. The McIDAS system was used to multiply the NS image by a normalization factor which matched it to the XR image with respect to exposure. After background subtraction from both images, the XR image was divided by the aligned normalized NS image; the resultant ratio image was multiplied by an enhancement factor and colored according to pixel brightness. Polypeptides with a high or low ratio relative to the average ratio stood out clearly in contrasting colors on the ratio image and could be detected by visual inspection. The actual XR/NS density ratios of these highlighted spots could then be quantitated by spot integration. To test the method, XR/NS density ratios were measured for 34 spots on each of the 4 films, sets and the spot ratios compared to the spot appearances on the ratio image. With the exception of a few false positives, agreement was good. Several polypeptides were found to be increased by 25–50 % relative to the average ratio while one polypeptide of molecular weight 50 000, approximate pI 5.5, was increas

ISSN:0173-0835    

DOI:10.1002/elps.1150030610

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractIn studies of genetic diseases such as Duchenne muscular dystrophy (DMD), two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) provides an indispensable tool for studying protein changes. For optimum use of 2‐D PAGE it is important to maximize the resolution that can be obtained. In this study we have developed a system using flat‐bed isoelectric focusing (IEF) with gels bound to a plastic backing for the first‐dimension separation. This system, when compared to the conventional rod IEF system, was found to yield good resolution of both the acidic and basic proteins, which are normally separated using non‐equilibrium systems. We have also used the principals of gradient engineering in both the IEF and sodium dodecyl sulfate (SDS)‐PAGE dimensions to maximize the gel area used for the separations. Mixtures of various commercial carrier ampholytes were also used and were found to further enhan

ISSN:0173-0835    

DOI:10.1002/elps.1150030611

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY