摘要:

AbstractWe have studied the separation of large (80–300 kbp) supercoiled (SC) DNA in conventional agarose gel electrophoresis, field inversion gel electrophoresis (FIGE) and pulsed field gel electrophoresis (PFGE). DNA migration was measured under a variety of electrophoretic conditions including different switch times, temperatures, agarose concentrations, and voltage gradients. The migration of SC DNA was found to be inversely proportional to its molecular weight in the three electrophoresis systems tested. In conventional agarose electrophoresis, voltage gradient was found to be the determining parameter in the separation of SC DNA. Unlike large linear DNAs, the migration of SC DNA was found to be independent of switch time in PFGE and FIGE. Broad DNA bands were observed in prolonged FIGE runs. In addition, we have also studied the migration of open‐circular (OC) DNA (80 and 100 kbp) in pulsed field gel electrophoresis. Eighty kbp OC DNA can migrate into agarose gels under certain pulsed field conditions whereas 100 kbp OC DNA was trapped at the wells. Based on electrophoretic conditions described in this report, we can determine the size of bacterial artificial chromosome (BAC) clones without restriction enzyme digestion and have enriched the percentage of larger size clones in BAC clon

ISSN:0173-0835    

DOI:10.1002/elps.1150160102

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

年代:1995

数据来源: WILEY

摘要:

AbstractUsing a newly developed temperature‐gradient gel electrophoresis apparatus, mutations in the K‐rasoncogene andp53tumor supressor gene were analyzed for single‐strand conformation polymorphism (SSCP). The mobilities of single‐stranded DNAs, carrying various mutations, change – depending on the gel temperature during electrophoresis. Therefore, temperature‐gradient single‐strand conformation polymorphism (TG‐SSCP) analysis can provide useful information concerning the optimum temperature for SSCP and may also be used to screen for various mutations or polymorphisms in a single elec

ISSN:0173-0835    

DOI:10.1002/elps.1150160103

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe Ogston‐Morris‐Rodbard‐Chrambach model (OMRCM) of gel electrophoresis assumes that the mobility μ of charged particles is proportional to the fractional volume (f) of the gel that is available to them. If the gel is random, as described by Ogston, the (semi‐log) Ferguson plot is the method of choice for analyzing experimental data since it permits an estimate of the gel's mean pore size to be made. However, the Ferguson plot is rarely linear; this is usually “explained” by the deformation of the anisotropy of the particle, the nonrandom or variable architecture or the gel, or the onset of some other migration mechanism. Many authors have refined this model, but the original assumption that μ ∝fhas not been seriously examined. Also, the model says nothing of the effect of the field intensity, the connectivity of the gel pores, nor anything about the diffusion coefficient. We have developed a Monte‐Carlo computer simulation algorithm to study the electrophoretic sieving of simple particles in gels. In this brief communication, we report important preliminary results which indicate that the basic assumptions of the OMRCM are wrong. We use a two‐dimensional periodic gel since the OMRCM becomes trivial in this case. Our results show that the relationship betweenfand μ is not the one assumed by the OMRCM. Moreover, we find that the Einstein relation between the diffusion coefficient and the mobility is not valid. This is due to the fact that the particles do not have a uniform probability of visiting the various sites that are available to them. We thus conclude that the Ferguson plot is intrinsically nonlinear; the curvature of the plot is, in fact, related to the intensity of the electric field as well as to the degree of randomne

ISSN:0173-0835    

DOI:10.1002/elps.1150160104

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

年代:1995

数据来源: WILEY

摘要:

AbstractAn informativity index for multilocus DNA fingerprints,r, was developed. It is based upon the Shannon information that a paternal DNA fingerprint conveys about the offspring phenotype pattern. Both simulation and empirical data reveal that the indexris strongly correlated with the mean log likelihood ratio (paternityvs. nonpaternity) expected in trio cases of true paternity. Sincercan be estimated from DNA fingerprints of unrelated individuals in advance, it will provide an easy means to assess the potential utility of a given probe/enzyme combination in kinship testing.

ISSN:0173-0835    

DOI:10.1002/elps.1150160105

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

年代:1995

数据来源: WILEY

摘要:

AbstractA set of commercially available peptides suitable for use as standards in denaturing isoelectric focusing (IEF) is described. The peptidesN‐procalcitonin fragment 1–57 (pI3.98), Gln11‐amyloid β‐protein fragment 1–28 (pI5.76), gastric inhibitory polypeptide (pI7.14), parathyroid hormone fragment 1–34 (pI8.64) and human β‐endorphin (pI9.49) can be focused to their isoelectric point in the presence of 8Murea and 2% Nonidet P–40, and subsequently fixed and stained in polyacrylamide gels. The peptides give a linear standard curve in close agreement with a slope determined with a surface pH electrode. Under the same conditions some proteins focus to positions significantly at odds with their theoretical isoelectric point. The origins of these discrepancies and the implications for the determination of isoelectric points of unknown proteins by denaturing

ISSN:0173-0835    

DOI:10.1002/elps.1150160106

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe pyrogallol red protein assay (Clinical Chemistry1986,32, 1551–1554) is based upon formation of a blue protein‐dye complex in the presence of molybdate under acidic conditions. However, centrifugation of the assay mixture results in loss of color yield and precipitation of the protein‐dye complex which can be recovered and resolubilized to achieve protein concentration prior to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The method has been evaluated relative to trichloroacetic acid (TCA) precipitation for recovery and electrophoresis of commercial protein and peptide molecular weight markers. Precipitation with pyrogallol red‐molybdate (PRM) gives better and more uniform recovery of both proteins and peptides as compared to TCA. The lower limit of PRM precipitation is similar to TCA and corresponds to 1 μg protein per mL assay mixture. This is equivalent to 100 μL of 10 μg/mL protein using the standard protein assay or 1 μg/mL protein using a modified assay incorporating a fivefold concentrate of the dye reagent. Application of the method is demonstrated by concentration of urinary proteins. The method is simple and economic and useful for conserving trace amounts of precious sample as it allows recovery of protein for electrophoresis following

ISSN:0173-0835    

DOI:10.1002/elps.1150160107

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

年代:1995

数据来源: WILEY

摘要:

AbstractElectrophoretic conditions including electrode and gel buffers, acrylamide concentration, use of stacking gels, voltage, current, and run time were investigated in order to produce isozyme bands of high resolution which would facilitate densitometric quantification of enzyme activity following polyacrylamide gel electrophoresis (PAGE). Electrode buffers which provided optimal conditions for gels stained for the isozymes of malate dehydrogenase (MDH), 6‐phosphogluconate dehydrogenase (6‐PGD), phosphoglucose isomerase (PGI), and shikimate dehydrogenase (SkDH) were 0.02MTris‐glycine, pH 8.5, 0.1Msodium borate, pH 6.0, 0.1Msodium borate, pH 8.7, and 0.07Msodium borate, pH 7.0, respectively. A 0.5MTris‐HCl, pH 7.5, gel buffer was optimal for gels stained for the isozymes of 6‐PGD, PGI and SkDH. A 0.5MTris‐HCl, pH 8.5, gel buffer was best for gels stained for MDH. Stacking gels were found to be detrimental to enzyme activity and showed no improvement in resolution for any of the enzymes. Acrylamide concentration for gels stained for MDH were 8.7%, gels stained for 6‐PGD and PGI were 7.5%, while gels stained for SkDH had an acrylamide concentration of 5.0%. Higher concentrations above these levels caused a reduction and in some cases loss of band activity, while below this concentration there was a decrease in band resolution. Gels stained for MDH yielded best results when run for 6.5 h at a constant current of 5 mA/gel and an initial voltage of 40 V, gels stained for 6‐PGD were best after 10 h at an initial current of 8 mA/gel and a constant voltage of 140 V, gels stained for PGI were run for 22 h at an initial current of 9 mA/gel and a constant voltage of 34 V and gels stained for SkDH were run for 10 h at an initial current of 5 mA/gel and a constant voltage of 60 V. Triscitrate buffers used widely inCitrusand other taxons on both starch and polyacrylamide gels were found to be unsatisfactory. Higher molarity buffers with lower current and longer run times were found to provide superior resolution and band separation in comparison to lower molarity buffers with higher current and shorter run times. Zones of activity previously reported inCitrusbut not in mandarin cultivars were revealed for both MDH and PGI. Our interpretation of the alleles for SkDH and 6‐PGD were not in agreement with those previously reported for the cultivars studied. These electrophoretic conditions provide isozyme bands of high resolution on PAGE, which will be suitable for densi

ISSN:0173-0835    

DOI:10.1002/elps.1150160108

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

年代:1995

数据来源: WILEY

摘要:

AbstractA rapid and sensitive method of detecting pectinesterase activity following electrophoresis or isoelectric focusing in polyacrylamide gels is described. The method uses ruthenium red and requires no addition of substrate when making the gels, thus obviating direct enzyme‐substrate contact during electrophoresis. Because of its versatility, the method can be used in a wide variety of applications, such as plant and microbial taxonomy, enzyme purification and characterization, or as an analytical method in fresh and processed plant technolog

ISSN:0173-0835    

DOI:10.1002/elps.1150160109

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

年代:1995

数据来源: WILEY

摘要:

AbstractA new method for the visualization of proteolytic activity in cell culture supernatant fromPseudomonas lundensisafter sodium dodecyl sulfate (SDS) – gel electrophoresis is described. Following conventional electrophoresis, the gel is washed in a methanol‐containing buffer to facilitate partial removal of SDS. After incubation with 0.5% casein the gel is stained for protein with Coomassie Brilliant Blue R‐250. Bands with proteolytic activity appear as clear areas in the gel against a blue‐stained background. Molecular weight standards electrophoresed in the same gel stain more intensely than the background and allow determination of the molecular weights of the proteolytic components. The sensitivity of post‐electrophoretic reactivation in SDS‐gels was determined using trypsin as standard. A slight modification of the technique allowed detection of proteolytic activity in nondenaturing and in isoelectric fo

ISSN:0173-0835    

DOI:10.1002/elps.1150160110

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

年代:1995

数据来源: WILEY

摘要:

AbstractAnions commonly found in biological fluids such as chloride, citrate, phosphate, bicarbonate, lactate, aspartate and glutamate, were determined within 60 s by means of capillary zone electrophoresis and indirect UV detection using 10 mMof chromate, pH 8.0, as both carrier electrolyte and chromophore. The lower mass detection limit was 0.5 pmol. In order to avoid a gradual shift of the pH and, in consequence, drifting ionic mobilities due to the formation of hydroxyl ions at the cathode, 1 mMof 5,5‐diethylbarbiturate was added to the background electrolyte. Moreover, fused silica capillaries were coated with a 0.0003% solution of hexadimethrine bromide for two minutes between runs to reverse electroendosmotic flow which otherwise would counteract the electrophoretic migration of the anions and, hence, increase time of analysis. It is concluded that charge‐reversed capillary zone electrophoresis in concert with indirect UV detection is superior to ion chromatography and conductivity detection in the determination of inorganic and organic anions in biological fluids both with regard to selectivity, sensitivity and speed of analysisPresented in part at the International Ion Chromatography Symposium, Linz, Austria, September 21–24,

ISSN:0173-0835    

DOI:10.1002/elps.1150160111

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

年代:1995

数据来源: WILEY