摘要:

AbstractWe have designed and constructed a computer‐controlled, fully automated system for Southern‐type nucleic acid hybridization analysis. Restriction enzyme digests of DNA are placed into sample wells of the gel contained on a nine‐fingered plastic frame. The32‐labeled probe is loaded into the hybridization chamber. Instructions for all the subsequent steps in the fully automated process are specified by the operator's answers to questions which appear on the computer screen at the start of the experiment. The system performs horizontal submarine electrophoresis. An adjustable endpoint detector concludes electrophoresis. Automatic voltage/temperature feedback control maintains maximum allowable voltage while keeping the temperature constant. Following electrophoresis a robot arm moves the gel frame from station to station. The system then affixes the separated fragments to a solid phase matrix, denatures, neutralizes, hybridizes, washes, dries and detects the32P according to the specifications preprogrammed by the operator. The results, printed out by the computer, give a plot of radioactivity versus distance from the origin for each of the nine simultaneous hybridi

ISSN:0173-0835    

DOI:10.1002/elps.1150080602

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

年代:1987

数据来源: WILEY

摘要:

AbstractNegatively charged polystyrene latex particles with electron microscopically determined radii of 22.4, 26.9, 27.6, 43.5, 44.2 and 59.4 nm (nominal radii provided by the manufacturer of 28.5 to 75 nm) were found to be applicable as size standards for the quantitative agarose gel electrophoresis of viruses and cellular particles in that size range. In electrophoresis at pH 6.5, 0.03Mionic strength in presence of 10 mM3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate (CHAPS), the polystyrene particles exhibit non‐linear, reproducibly convex‐sigmoidal Ferguson plots. This curve shape is compatible with a model specifying a dependence of gel fiber dimensions and of particle size on the gel concentration. Based on a standardization of the agarose fiber by the polystyrene latex particles, computer simulation methods were used to determine the effective radii (without added Ca++) of turnip crinkle virus (TCV), hibiscus chlorotic ringspot virus (HCRSV) and pelargonium flowerbreak virus (PFBV) as 29.4, 24.2 and 22.1 nm, and their net charges as free mobilities, μoof 9.89, 10.87 and 16.20 · 10−5cm2/s/V, respectively. Within the size range of the standards, a simplified procedure for determination of particle size from curved Ferguson plots appears applicable as a first approximation (Zwaan method). When electrophoresis was conducted in the presence of 5 mMCa++, the effective particle radii of TCV, HCRSV and PFBV were reduced to 12.8, 20.3 and 18.3 nm, respectively. This compares with previous electron microscopic estimates of approximately 15 nm for TCV, HCRSV and PFBV. The observed increase of effective particle radii in the absence of Ca++corroborates the particle swelling and/or shape change at pH's ≥ 5.5 in the absence of Ca++which is known to occur with

ISSN:0173-0835    

DOI:10.1002/elps.1150080603

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

年代:1987

数据来源: WILEY

摘要:

AbstractTwo procedures of computer simulation of the electrophoretic migration of a particle through agarose gel are described which allow for: (a) characterization of gel fiber dimensions as a function of gel concentration (gel standardization), (b) determination of particle radius and the dynamics of apparent particle compressibility during passage through the standardized gel, and (c) estimation of the net charge density of a particle by calculating its mobility at 0 % gel concentration. The common model underlying these simulations is based on the extended Ogston theory which probabilistically describes the migration of a particle through a random network of inert and non‐flexible fibers in terms of a “random space walk”. The first procedure, applicable to relatively rigid particles such as bacteriophages, standardizes the gel fiber on the basis of mobility values (cm/s)/(V/cm) at several gel concentrations of a single, or several, bacteriophages of known radius. Mobilities of an unknown bacteriophage are then used to simulate its physical properties. The second method, applicable to relatively non‐rigid particles such as plant viruses, uses 7 polystyrene particles of known radius to standardize the gel fiber, followed by simulation of virus properties on the basis of their mobilities at several gel concentrations. The techniques described are most appropriate for deriving physical properties of particles from their nonlinear plots of log (mobility)vs.gel concentration (Ferguson plots). They have the virtue of yielding the properties of native, hydrated gel fibers and pa

ISSN:0173-0835    

DOI:10.1002/elps.1150080604

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

年代:1987

数据来源: WILEY

摘要:

AbstractThree 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/cm2of 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 el

ISSN:0173-0835    

DOI:10.1002/elps.1150080605

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

年代:1987

数据来源: WILEY

摘要:

AbstractAll four sequenced variants of human plasma transthyretin (TTR) with different substitutions of electrically neutral amino acids and associated with familial amyloidotic polyneuropathy (i. e.TTR(Met30), TTR(Ala60), TTR(Tyr77) and TTR(Ser84)) have been separated from the normal TTR monomer by hybrid isoelectric focusing (HIEF) under denaturing conditions. The pIdifferences from the normal monomer were between 0.0025 and 0.005 pH units corresponding to 0.7–1.4 % of a full charge unit difference. The detectability of these variants by HIEF under denaturing conditions (7Murea, 50 mMdithiothreitol, 2 % Triton X–100) was explained assuming interaction of the amino acids at the substitution site with a charged amino acid in its close vicinity. It is proposed to distinguish a high from a low level of resolution for HIEF under denaturing conditions to underline the fact that the studied variants could only be detected by extreme flattening of the pH gradient. To provide protection against modification by the laboratory atmosphere the entire gel, including the electrode wicks and sample application site, was covered with a layer of paraffin

ISSN:0173-0835    

DOI:10.1002/elps.1150080606

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

年代:1987

数据来源: WILEY

摘要:

AbstractA simple method of immobilizing protein on cellulose acetate by pretreating the films with methacryloxypropyltrimethoxysilane is presented. When a print is taken with such a substrate film the separating gel is not contaminated by the immobilized substrate, thus permitting location of focused proteins by means of silver stamina Dried films with immobilized substrate can be stored for several months before use Detection of alkaline and acid proteases is possible with a sensitivity of 0.3 ng/mm2.

ISSN:0173-0835    

DOI:10.1002/elps.1150080607

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

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080608

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

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080601

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

年代:1987

数据来源: WILEY