ISSN:0173-0835    

DOI:10.1002/elps.1150010302

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

年代:1980

数据来源: WILEY

摘要:

AbstractAffino‐titration curves, a variant of conventional affino‐electrophoresis techniques, allow the simultaneous determination of protein‐ligand dissociation constants (Kd) and of their pH‐dependence in the pH range 3–10. If the ligand is a macromolecule, it is simply entrapped in the gel matrix; if it is a small molecule, it is covalently bound to the gel fibers. When a titration curve of a protein is run in presence of increasing amounts of ligand in the gel, the progressively decreasing mobilities, when plotted against the ligand molarity in the gel, can be used to calculate Kd's at any pH value. This technique has been used to measure the dissociation constants of glycogen with phosphorylases a and b, and their pH

ISSN:0173-0835    

DOI:10.1002/elps.1150010303

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

年代:1980

数据来源: WILEY

摘要:

AbstractA maximum utilization of the high resolving power of isoelectric focusing requires that the focused zones are both planar and perpendicular to the direction of focusing. This paper reports on extensive investigations carried out with a rectangular quartz column with provisions for direct photography of zonal shapes. The experiments have been performed in density gradients, but the conclusions may be generalized to other stabilizing media. It is demonstrated that distorted zones are formed if a second pH gradient, perpendicular to the direction of focusing, originates at one end of the column. The generation of such a pH gradient may have different causes: erroneous loading of the column, too high an initial power input, presence of salts of weak electrolytes in the sample, or unsuitable positioning of the electrodes. The effects of the second pH gradient on the shapes of the focused carrier‐ampholyte and protein zones are interpreted on the basis of the mechanism creating the natural axial pH gradient. The stability of the disturbed system is discussed in terms of the diffusional, electrical, and convective mass flows of its component

ISSN:0173-0835    

DOI:10.1002/elps.1150010304

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

年代:1980

数据来源: WILEY

摘要:

AbstractA natural pH gradient can be formed on polyacrylamide gel using either two (glutamic acid and lysine only) or three (glutamic acid, histidine and lysine) aminoacids as the sole carrier ampholyte species. In the two component system, glutamic acid and lysine focus as single peaks adjacent (anodically and cathodically, respectively) to the steep, linear center of the step‐function pH gradient. Histidine, in the three component system, focuses at the center of the pH gradient and separates the other two aminoacids. Each aminoacid electrofocuses at its pI. Hemoglobin electrofocuses as a sharp band in both systems and, in the two component system, remains focused at its pI for 200 h. A major conductance gap forms at the gel center in both systems with progressing time of electrofocusing; a minor and transient conductance gap develops in the glass tube just above the ge

ISSN:0173-0835    

DOI:10.1002/elps.1150010305

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

年代:1980

数据来源: WILEY

摘要:

AbstractMethods of two‐dimensional protein fractionation have been analysed for their suitability for the analysis of heterogeneous ribonucleoprotein (hnRNP) particles. Nonequilibrium pH gradient electrophoresis gives the best overall fractionation and reveals that the dominant “core” proteins possess charge heterogeneity. For the analysis of the minor components of the particles, it may be preferable to overload isoelectric focusing gels and allow the basic core proteins, which comprise 70% of the total protein, to migrate off the gel. This does, however, result in the loss of some basic minor polypep

ISSN:0173-0835    

DOI:10.1002/elps.1150010306

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

年代:1980

数据来源: WILEY

摘要:

AbstractThe two‐dimensional (iso‐dalt) electrophoresis system for protein separation has been modified to include the use of flat‐bed agarose isoelectric focusing in the first dimension, horizontal SDS‐electrophoresis in an exponential acrylamide gradient in the second dimension, molecular weight standards run side by side with the sample in the SDS‐polyacrylamide gel, and radiolabeled proteins as internal standards. Direct determination of the pH gradient after the first dimension and of molecular weights after the second dimension permits more accurate measurement of the protein spots in both the x and y axes, thus improving the confidence limits of the technique. The reproducibility of molecular weight determinations was calculated for eight different proteins in six different gels, with an overall standard deviation of 3.02%. Incorporation of internal standards is useful in identifying and confirming protein patterns. Examples of the application of this technique to the separation of specific plasma proteins are

ISSN:0173-0835    

DOI:10.1002/elps.1150010307

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

年代:1980

数据来源: WILEY

摘要:

AbstractThe present paper gives a description of a procedure for isolation of pepsinogen I from human urine. The initial steps are concentration by batch adsorption to DEAE‐Sephadex, desorption by addition of NaCl, and DEAE‐chromatography. The final step is preparative isotachophoresis with Ampholine carrier ampholytes as spacers. Exact visualization of the fractionations is obtained by fused rocket immunoelectrophoresis. The procedure provides pepsinogen I of high purity as shown by crossed immunoelectrophoresis, agar gel electrophoresis and N‐terminal aminoacid analysis. The yield of the procedure is 0.05–0.1 mg pepsinogen I per lite

ISSN:0173-0835    

DOI:10.1002/elps.1150010308

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

年代:1980

数据来源: WILEY

摘要:

AbstractA continuous‐flow isoelectric focusing (IEF) system for protein fractionation, using a non‐commercial separation chamber and carrier ampholytes synthesized in the laboratory, is described. The resolving power and the sample‐loading capacity have been tested by the fractionation of hemoglobin mixtures. Hb A2and HbA (ΔpI 0.4) were separated with a sample loading of 4 g of hemoglobin per day. The maximal temperature in the gel was 5 °C, the average field strength 30 V/cm and the residence time 6 h. The continuous‐flow IEF system was further used as a preliminary step in the purification of choline acetyltransferase (EC. 3.2.1.6) from human placenta. The results obtained with choline acetyltransferase indicated that continuous‐flow IEF can be used for the purification of enzymes when other preparative IEF systems are not effective, particularly when the sample is present in l

ISSN:0173-0835    

DOI:10.1002/elps.1150010309

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

年代:1980

数据来源: WILEY

摘要:

AbstractThe hydrophobic protein comprising the gas vacuoles of a variety of procaryotic microorganisms is insoluble in detergents (e.g.sodium dodecyl sulfate) and cannot be electrophoresed in detergent‐containing systems. The protein is solubilized by mixtures of phenol‐acetic acid‐urea (PAU) and the development of a slab gel system containing PAU which successfully separates the gas vacuole protein is described. In the system, phenol is polymerized directly into the gel, and the high background staining which results can be minimized by the proper choice of gel thickness, acrylamide/Bis ratio, and polymerization conditions. There is a direct relationship between the electrophoretic mobility and molecular weight of protein standards in the PAU system; however, several proteins including histones H2, H3, H4and bovine serum albumin have a faster rate of gration than expected. It has been possible to separate and identify the gas vacuole protein fromHalobacterium halobium, Halobacterium salinariumstrain 5, and the two species of cyanobacteria,Anabaena flos‐aquaeandMicrocystis aeruginosa. Mobility of the gas vacuole proteins in the four species are consistent with molecular weights of 16 800, 16 800, 14 700 and 15 700 respectively. Gels produced using the PAU system can be successfully fluoro

ISSN:0173-0835    

DOI:10.1002/elps.1150010310

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

年代:1980

数据来源: WILEY

摘要:

AbstractThe distribution pattern of3H‐labeled proteins in slab gels has been recorded with two instruments: a thin‐layer scanner and a Betacamera, both of which have been used with success previously in monitoring paper and thin‐layer chromatograms. Individual radioactive protein bands may be localized with both instruments although they differ markedly in sensitivity. The scanner is relatively insensitive and thus time‐consuming, since each lane of the gel has to be recorded separately. In contrast, with the Betacamera the whole area of the slab gel could be monitored simultaneously. The extraordinarily high sensitivity of the Betacamera in detecting3H‐compounds via the spark chamber proved to be of great advantage since we intended to isolate preparatively specific proteins from the gel. The “betagram” technique has a very low detection limit and is thus less time‐consuming than fluorography. However, the resolution of the “betagram” is limited due to the number of wires in the spark chamber, and does not reach the quality attaine

ISSN:0173-0835    

DOI:10.1002/elps.1150010311

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

年代:1980

数据来源: WILEY