摘要:

AbstractComputer simulations of the concentration profiles of simple biprotic ampholytes with Δ pKs 1, 2 and 3, on immobilized pH gradients (IPG) at extreme pH values (pH 3–4 and pH 10–11) show markedly skewed steady‐state profiles with increasing kurtosis at higher Δ pKvalues. Across neutrality all the peaks are symmetric irrespective of their Δ pKvalues, but they show very high contribution to the conductivity of the background gel and significant alteration of the local buffering capacity. The problems of skewness, due to the exponential conductivity profiles at low and high pHs, and of gel burning due to a strong electroosmotic flow generated by the net charges in the gel matrix, also at low and high pHs, are solved by incorporating in the IPG gel a strong viscosity gradient. This is generated by a gradient of linear polyacrylamide which is trapped in the gel by the polymerization

ISSN:0173-0835    

DOI:10.1002/elps.1150070202

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe effcient and rapid technique of ultrathin‐layer isoelectric focusing on 50 μm polyacrylamide gels was used for determination of heat‐induced changes of whey proteins (β‐lactoglobulin A and B as well as α‐lactalbumin) and a method for quantitative densitometry was developed. Improved reproducibility could be achieved by (i) introducing an internal standard (carbonic anhydrase), allowing for compensation for minor irregularities in gel composition, sample application or staining, (ii) running each sample in triplicate, and (iii) multiple scanning of each sample at different positions of the focusing track. Coefficients of variation of the integrated peak areas were approximately 3 % for β‐lactoglobulin A and B and about 6 % for α‐lactalbumin. Skim milk was heated in a pilot heating plant especially designed for reaction kinetic studies. Using the initial content of native whey proteins in each milk sample as a reference value, degrees of denaturation of more than 90 % could be determined for each of the three protein fractions. Results obtained from reaction kinetic studies revealed that denaturation of α‐lactalbumin was a f

ISSN:0173-0835    

DOI:10.1002/elps.1150070203

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

年代:1986

数据来源: WILEY

摘要:

AbstractWe have prepared a standard of human myoglobin by flat bed isoelectric focusing, to be used in immunological and chemical methods of measurement in clinical biology. Myoglobin was extracted from human psoas or myocardium and then purified by preparative isoelectric focusing in a granulated gel. The purified myoglobin was examined by analytical isoelectric focusing, high performance liquid chromatography (HPLC), immunoelectrophoresis, and two‐dimensional gel electrophoresis. Myoglobin was quantified by three methods: (i) Drabkin's method, and by the determination of iron using (ii) the bathophenanthroline method, and (iii) the atomic emission method. There was good correlation between all three methods. Purified myoglobin was stabilized by conversion to the cyanmetmyglobin form, and stored until use at 4 °C (± 2 °C). or at – 20 °C in the liquid state after addition of ethylene

ISSN:0173-0835    

DOI:10.1002/elps.1150070204

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe reproducibility of protein migration was studied in three electrophoretic systems: sodium dodecyl‐sulfate electrophoresis on polyacrylamide gradient, isoelectric focusing on immobilized pH gradients and two‐dimensional charge‐size separations. The gradient gels were poured either with a two‐vessel apparatus or with a set of computer‐driven burettes, as described by Altland and Altland (Electrophoresis1984,5, 143–147). Standard deviation on band position was cut by 20–50% with the latter approach in comparison with the former. Typical results were: sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, 0.6 mm (over a total length of 135 mm); immobilized pH gradients, 0.6 mm (for 179 mm length); two‐dimensional separations, 0.8 mm (χ‐ax

ISSN:0173-0835    

DOI:10.1002/elps.1150070205

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

年代:1986

数据来源: WILEY

摘要:

AbstractTwo‐dimensional electrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by silver staining or specific immunoblotting analysis, was applied to the characterization of gamma‐glutamyltransferase (GGT, EC 2.3.2.2.) in rat tissues. We confirmed that purified kidney GGT is a dimer composed of two non‐identical subunits with molecular masses of about 50 and 30 kDa. Both the light and the heavy subunits were separated into 6 and 2 protein bands, respectively. Antibody fractions against the 30 and 50 kDa subunits, purified by immunoaffinity from a whole antiserum directed to the rat kidney enzyme, recognized their corresponding subunits and did not cross‐react with each other. Studies of the reactivity of these antibodies towards GGTs from kidney and liver homogenates revealed an intraspecies dissimilarity in the molecular architecture of the kidney and liver GGTs, especially concerning the 30 kDa subunit. Following phenobarbital treatment of animals we observed an increase in immunoreactive GGT, accompanied by the appearance of additional polypeptides with more basic isoelectric points and higher molec

ISSN:0173-0835    

DOI:10.1002/elps.1150070206

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

年代:1986

数据来源: WILEY

摘要:

AbstractWith the aid of two‐dimensional electrophoresis, the immunoglobulins (Ig) in cerebrospinal fluid (CSF) were separated into heavy and light chains and visualized with silver staining. CSF from 36 patients with no abnormal immunologic activity in the central nervous system show a constant pattern of light chain subfractions, however varying strongly in their respective quantities. In 40 patients with inflammatory diseases such as confirmed multiple sclerosis (MS) (n = 10), neurosyphilis (n = 6), lymphocytic meningoradiculitis (n = 3), novel immunoglobulins appear and form a so‐called oligoclonal zone in the light chain region. By comparing various oligoclonal zones, differences were found between the arrangement of the novel Ig light chains and the polyclonal light chain background. In the region of the polyclonal light chain background certain protein fractions appear which are non‐immunoglobulin and which are not found in serum. The protein fraction at the isoelectric point of 7.8 appears in 9 out of 10 confirmed MS cases, but in none of the 6 neurosyphilis and 3 lymphocytic miningoradicultis cases. In one case of neurosyphilis with an extremely high gamma‐globulin content in CSF we found an oligoclonal zone in the heavy chain

ISSN:0173-0835    

DOI:10.1002/elps.1150070207

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

年代:1986

数据来源: WILEY

摘要:

AbstractA method for the detection of disulfide bonded polypeptide components in protein mixtures is described. During the electrophoresis of proteins in sodium dodecyl sulfate‐polyacrylamide slab gels, the migration is interrupted and advantage is taken of the rapid diffusion of added 2‐mercaptoethanol into lanes without reductant. In this paper, the results of applying this procedure to proteins known to contain only inter‐chain (α2‐macroglobulin, fibrinogen, type III collagen) and intrachain disulfides (bovine serum albumin, alkaline phosphatase) as well as to crude extracts are

ISSN:0173-0835    

DOI:10.1002/elps.1150070208

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

年代:1986

数据来源: WILEY

摘要:

AbstractA common problem, when using Silane A 174 to bind polyacrylamide gels to supporting glass plates, is that the gel also binds to the cover glass in the gel casting frame. This can be avoided by using a low concentration of Silane A 174 and by rinsing the supporting glass plate with acetone after the Silane A 174 treatment.

ISSN:0173-0835    

DOI:10.1002/elps.1150070209

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

年代:1986

数据来源: WILEY

摘要:

AbstractA method is described for direct analysis of the reaction mixtures of the production of the herbicide glyphosate and the growth regulator glyphosine by capillary isotachophoresis providing simultaneous determination of the products N‐phosphonomethyl‐glycine and N.N‐bis(phosphonomethyl)glycine as well as of all important reaction components ‐ sulfuric, formic, phosphorous, phosphoric, hydroxyacetic, nitrilotriacetic, iminodiacetic and N‐phosphonomethyliminodiacetic acids and glycine. The method is suitable for the analyses both of the final products and of mother liquors, where the maximum ratio of the concentrations ranges within 1:100. The time of analysis is dependent on the sample composition and ranges within 5–20 min. relative standard deviation of a single determination being less th

ISSN:0173-0835    

DOI:10.1002/elps.1150070210

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

年代:1986

数据来源: WILEY

摘要:

AbstractA new method for analytical isoelectric focusing of the benzodiazepine receptor in calf brain is described. Brain membranes are photoaffinity labeled with a specific radioligand, solubilized in 9 M urea and separated on 150 μm ultrathin polyacrylamide gels containing 8 M urea. The method combines high resolution with speed and versatility. It may be generally applicable for isoelectric focusing of integral membrane proteins in genetic studies

ISSN:0173-0835    

DOI:10.1002/elps.1150070211

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

年代:1986

数据来源: WILEY