摘要:

AbstractA widened use of isoelectric focusing for preparation of large amounts of pure proteins is highly desirable but is severely hampered,inter aliaby 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 pr

ISSN:0173-0835    

DOI:10.1002/elps.1150020402

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

年代:1981

数据来源: WILEY

摘要:

AbstractDetails 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−1per 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

ISSN:0173-0835    

DOI:10.1002/elps.1150020403

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

年代:1981

数据来源: WILEY

摘要:

AbstractPolymerization 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 highl

ISSN:0173-0835    

DOI:10.1002/elps.1150020404

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

年代:1981

数据来源: WILEY

摘要:

AbstractThe 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

ISSN:0173-0835    

DOI:10.1002/elps.1150020405

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

年代:1981

数据来源: WILEY

摘要:

AbstractAn 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 [

ISSN:0173-0835    

DOI:10.1002/elps.1150020406

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

年代:1981

数据来源: WILEY

摘要:

AbstractThe 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

ISSN:0173-0835    

DOI:10.1002/elps.1150020407

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

年代:1981

数据来源: WILEY

摘要:

AbstractA 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 heteropolyme

ISSN:0173-0835    

DOI:10.1002/elps.1150020408

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

年代:1981

数据来源: WILEY

摘要:

AbstractWhen 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

ISSN:0173-0835    

DOI:10.1002/elps.1150020409

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

年代:1981

数据来源: WILEY

摘要:

AbstractA 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‐equlib

ISSN:0173-0835    

DOI:10.1002/elps.1150020410

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

年代:1981

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150020411

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

年代:1981

数据来源: WILEY