ISSN:0173-0835    

DOI:10.1002/elps.1150111003

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

年代:1990

数据来源: WILEY

摘要:

AbstractTemperature‐gradient gel electrophoresis (TGGE) is a technique for studying the structural transitions of nucleic acids and proteins. A temperature gradient is formed in a horizontal slab gel perpendicular to the direction of the electric field. Whereas the principle of the TGGE method has previously been applied to proteins, we describe in this report the systematic optimization of TGGE as a routine technique for the quantitative analysis of conformational transitions in proteins. Using α‐amylase as an example we show the kinds of results which may be obtained from such measurements. Buffers suitable for use in gel electrophoresis were analyzed with respect to the dependence of their pH value upon temperature. The correct pH range for TGGE of a given protein is determined by electrophoretic titration curves. The protein bands are detected by silver and/or activity staining. The thermal denaturation of α‐amylase fromAspergillus oryzaeshowed a discontinuous transition into the denatured conformation, which exhibited much slower electrophoretic mobility. The discontinuity is due to an irreversible denaturation process under the gel conditions. The transition temperature was measured as a function of several parameters,e. g., the concentration of Ca++, dithiotreithol, urea and the pH value. The structural transition of α‐amylase is accompanied by a loss of enzymatic activity as determined by activity staining or by an activity assay carried out in solution. The structural transitions of two other α‐amylases fromBacillus subtilisandBacillus licheniformiswere also studied. The results show that the TGGE method is simple to perform and allows the analysis of conformational transitions of proteins in a wide variety of conditions. It is also possible to analyze the conformational stability of proteins in unpurified extracts if activity‐ or immuno‐tests are u

ISSN:0173-0835    

DOI:10.1002/elps.1150111004

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe principle and some applications of multiphasic electrophoresis with diversified spacers are described. The method combines previously published concepts of introducing highly diversified spacers into the stack of an isotachophoretic system with the theory of snow‐ and telescope‐electrophoresis. The link between this electrophoretic method and isotachophoresis on the one hand and monophasic zone electrophoresis on the other hand is exemplified. The importance of the nature of the spacers as well as of the pH and ions in the leading and terminating zone is illustrated. Details are given concerning the experimental set‐up. This electrophoretic technique could serve as an alternative to isoelectric foc

ISSN:0173-0835    

DOI:10.1002/elps.1150111005

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

年代:1990

数据来源: WILEY

摘要:

AbstractA method is described for detecting polymorphisms of cephalothorax and tail homegenates of 25 puerulus stagedPanulirus argusin phosphoglucomutase (PGM) a esterases. Isoelectric focusing in immobilized pH gradients was used. In the pH 6.0–8.0 interval for phosphoglucomutase and in the pH 3.5–5.0 and 4.2–4.9 rang for esterases both enzymes appeared as polymorphic band patterns. These could explained by one locus with 2 alleles for phosphoglucomutase and 3 loci with 2, 3 and 4 alleles for esterases. Esterases exhibit a more extensive polymorphism in immobilied pH gradients than in polyacrylamide gel electropho

ISSN:0173-0835    

DOI:10.1002/elps.1150111006

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

年代:1990

数据来源: WILEY

摘要:

AbstractA method is described for the separation and detection of highly alkaline IgG bands in unconcentrated cerebrospinal fluid (CSF). These bands are frequently found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system, particularly in the case of multiple sclerosis, and their detection is an important aid in clinical diagnosis. An isoelectric focusing technique using an immobilised pH gradient in polyacrylamide gel has been developed over the pH range 7–10, producing a linear and stable pH gradient with excellent resolution. After electrofocusing, the protein patterns were blotted onto polyvinylidene difluoride membranes and visualised using anti‐human IgG followed by an enzyme‐labelled second antibody. Blotting could be carried out by capillary diffusion for up to 16 h duration without any loss in resolution. Using this method, highly alkaline intrathecal IgG bands were found in the cerebrospinal fluid of all of the 14 multiple sclerosis patients. There were also 2 patients with alkaline IgG bands in their cerebrospinal fluid who were not diagnosed as multiple sclerosis. By contrast, no alkaline IgG bands with an isoelectric point (pI) greater than 8.6 were found in any of the serum samples studied (n= 50) from patients with various neurological disorders including multiple scle

ISSN:0173-0835    

DOI:10.1002/elps.1150111007

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

年代:1990

数据来源: WILEY

摘要:

AbstractUsing horizontal thin‐layer isoelectric focusing in polyacrylamide gels, we separated the isoenzymes of carboxylic esterase (EC 3.1.1.1) of cell extracts prepared from human hematopoietic cells. Isoenzyme bands were visualized by staining with naphthol ester as substrate and coupling to an azo dye. Staining intensities of isoenzymes were quantified by densitometric scanning. On isoelectric focusing in a pH 2–11 gradient, distinct esterase isoenzyme profiles could be discerned and correlated to various types of normal hematopoietic cells and their leukemic counterparts. One unique isoenzyme, termed monoband, could be clearly identified on the basis of its isoelectric point (pI6.0), its strong expression by normal and malignant monocytes and its complete and selective inhibition by sodium fluoride. This band was only found in monocytes of either normal or leukemic origin, but not in lymphoid or myeloid cells. The monocyte esterase could be inhibited by sodium fluoride whereas other isoenzyme bands were resistant to this inhibition. However, the specificity of this inhibitory reaction was relative, depending on the concentration of sodium fluoride. Compared with normal monocytes, leukemic monocytes often showed an overexpression of the mono‐bands. Dilution experiments established the distinct prominence of the mono‐band which could be detected among the other isoenzymes even when only 1% of the total cell population consisted of monocytes. Immature myeloid, but mono‐band negative leukemic cells whose arrest of differentiation can be overcome byin vitro12‐O‐tetradecanoylphorbol 13‐acetate‐promoted differentiation to more mature cells, could be induced to express the mono‐band which paralleled their maturation to monocytes. Isoelectric focusing of esterase isoenzymes represents a powerful tool for the isoenzymatic analysis of hematopoietic cells and an enzymological classifi

ISSN:0173-0835    

DOI:10.1002/elps.1150111008

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

年代:1990

数据来源: WILEY

摘要:

AbstractTo test seed lots of tomato F1 hybrid varieties for the presence of undesirable inbred seeds by electrophoresis, a method has been developed on the basis of ultrathin‐layer isoelectric focusing. The method is based on the genetic variation of the seed protein PRS‐1 which could be visualized by isoelectric focusing of a 5 mm NaCl‐soluble seed protein extract in a pH 6–9 gel followed by protein staining. Two genetic variants of the PRS‐1 protein, PRS‐1+and PRS‐11, were found among open‐pollinated varieties, as well as among F1 hybrid varieties. The isoelectric points (pI) of the PRS‐1 proteins are 7.1 and 6.1 for PRS‐1+and PRS‐11, respectively. The PRS‐1 protein is unique to seed tissue and is located primarily in the embryo. A genetic 1:2:1 segregation of the genePrs‐1among several F2 populations shows monogenic inheritance. Analysis of commercial F1 hybrid varieties from several seed companies indicated that thePrs‐11allele, in contrast to thePrs‐1+allele, is primarily present in gene pools of „Moneymaker type”︁ tomatoes. The described method is generally applicable to all tomato F1 varieties that are heterozygous for the genePrs‐1. With the described method one person can routinel

ISSN:0173-0835    

DOI:10.1002/elps.1150111009

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

年代:1990

数据来源: WILEY

摘要:

AbstractFrom the leaf tissue of healthy and blighted citrus trees 10–30 kDa soluble fractions were compared to find biochemical markers of tissue in the disease state. Using a non‐denaturing extrction technique coupled with ultrafiltration, a resulting 10–30 kDa healthy and citrus blight fraction was sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Two distinct and adjacent bands for blight at anMrof approximately 12 500 were separated. These bands were visible with Coomassie Brilliant Blue and silver stain but were negative to glycoprotein stains. An antiserum prepared against proteins isolated by preparative electrophoresis reacted only with the blight fractions and was distinctly different from healthy fractions when Western blotted. Only the gel region (Mr12 500–13 000) of citrus blight sources was positive to the antiserum when compared with disease and nondisease stress sources. Results indicate that identification of specific proteins may be a way to diagnose the onset of citrus blight prior to visible tr

ISSN:0173-0835    

DOI:10.1002/elps.1150111010

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

年代:1990

数据来源: WILEY

摘要:

AbstractA procedure which can detect subtype‐specific minor bands of factor B (BF) by polyacrylamide gel isoelectric focusing is presented. After zymosan‐mediated fragmentation of BF in serumviaalternative pathway for complement activation, serum samples are subjected to isoelectric focusing in a narrow pH range (4.2–4.9). The Ba fragments are detected by using immunoblotting. In addition to the previously reported minor bands with subtypic specificities, heterogeneities are observed in other minor band group, where a single minor band corresponds exclusively to a subtype in a regular combination with the previously announced subtypic patterns. A one‐to‐one correspondence of a single band to each subtype provides an unambiguous determination for three subtypic phenotypes deduced from the two divided BF*F alleles, BF*FA and BF*FB. An autosomal codominant heredity is confirmed through segregation analysis. A population survey reveals that four common alleles, BF*S, BF*FA, BF*FB, BF*Fb1, occur in a Japanese population and the former three alleles, except BF*Fb1, occur in a Cambodian population. The presence or absence of a single anodal minor band was found to be the only difference after neuraminidase treatment of FA and FB, implying that an amino acid substitution responsible for the FA–FB subtypic difference is involved in an additional acquisition in FA of an oligosaccharide unit with a charged

ISSN:0173-0835    

DOI:10.1002/elps.1150111011

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

年代:1990

数据来源: WILEY

摘要:

AbstractGenetic variation of orosomucoid (ORM) in the genusMacacawas investigated Plasma samples were subjected to isoelectric focusing in a pH range of 4–6.5, followed by immunoprinting with anti‐human ORM antibodies. A total of 25 allele were identified in 231 Asian macaques belonging to 13 species from 23 population and 22 members belonging to a family ofM. fascicularis.Family data presented evidence for a codominant mode of inheritance with multi‐alleles at a single auto somal locus. A population study revealed enormous intra‐ and interspecies variations. The heterozygosity values varied from 0.855 inM. fascicularis(Malaysia) to 0.000 inM. radiata(India),M. silenus(India) andM. arctoides(Ma

ISSN:0173-0835    

DOI:10.1002/elps.1150111012

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

年代:1990

数据来源: WILEY