ISSN:0173-0835    

DOI:10.1002/elps.1150101202

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

年代:1989

数据来源: WILEY

摘要:

AbstractA high resolution two‐dimensional affinity electrophoresis has been developed, using capillary isoelectric focusing as the first electrophoresis and slab gel affinity electrophoresis as second electrophoresis. By this method 1–2 μg of anti‐dinitrophenyl antibodies have been separated completely into several hundred homogeneous IgG spots. They are grouped into a number of families which are composed of several IgG spots of the same affinity to the hapten but of a different pI. It is suggested that each individual family is derived from one monoclonal antibody producing cel

ISSN:0173-0835    

DOI:10.1002/elps.1150101203

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe molecular heterogeneity of rabbit anti‐hapten antibodies has been investigated by two‐dimensional affinity electrophoresis (2D‐AEP). Anti‐dansyl and anti‐arsanilic diazo‐antibodies were separated into several hundred IgG spots as in the case of anti‐DNP antibodies. They were grouped into a number of monoclonal IgG families. At the beginning of immunization, IgG spots having low pIand low affinity were predominant but one or two weeks after immunization the IgG spots with high affinity and high pIincreased. After the second or third immunization, the 2D‐AEP patterns became stable and constant. Anti‐arsanilic diazo and anti‐DNP antibodies exhibited only weak cross‐reactivity with other aromatic haptens. In contrast, anti‐dansyl antibodies cross‐reacted to a considerable degree with DNP‐hapten. A few anti‐dansyl IgG families which have cross‐reactivity with DNP‐hapten were separated. Their apparent dissociation constants to the haptens and their affinity ratios were calculated from their 2D‐AEP pattern

ISSN:0173-0835    

DOI:10.1002/elps.1150101204

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

年代:1989

数据来源: WILEY

摘要:

AbstractBy means of two‐dimensional lectin affinity electrophoresis of human α‐fetoprotein (AFP) from different sources, AFP bands separated with erythroagglutinating phytohemagglutinin (E‐PHA) were further characterized with other lectins of known oligosaccharide specificities. The results with a cord serum AFP revealed that not only AFP‐P2 (E‐PHA‐nonreactive) but also AFP‐P4 and P5 (E‐PHA‐reactive) had affinities for Concanavalin A (Con A) andAllomyrina dichotomalectin (allo A), indicating that the cord serum AFP has nonbisected biantennary complex‐type oligosaccharides with the terminal galactose on Man α1 → 6 residue sialylated at the C‐6, but not C‐3, position. On the other hand, the results with a hepatoblastoma (HUH‐6 C1–5 cell line) AFP showed that not only AFP‐P5 but also AFP‐P1 (E‐PHA‐nonreactive) and P3 (E‐PHA‐less reactive) had Con A‐nonreactive AFP and that AFP‐P1 had AFP‐A1 (allo A‐nonreactive) and AFP‐A2 (allo A‐less reactive), and AFP‐P3 and P4 had AFP‐A1s (allo A‐nonreactive), as main components, in addition to the spots of cord serum AFP. Most of the E‐PHA‐dependent bands of AFP were further subdivided withLens culinarisagglutinin (LCA‐A) into LCA‐A‐reactive, weakly reactive and nonreactive spots. Similar results were obtained with AFP preparations from hepatocullular carcinomas and other malignancies, indicating that the bisected bi‐(or tri‐and tetra‐) antennary sugar chains with the exposed terminal galactose of the Man α 1 → 6 arm as well as those with the C‐3 sialylated galactose residues could be expressed in AFP upon malignant transformation. The two‐dimensional lectin affinity electroph

ISSN:0173-0835    

DOI:10.1002/elps.1150101205

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

年代:1989

数据来源: WILEY

摘要:

AbstractA model system for studies of mechanisms governing the alterations of glycosylation of plasma glycoproteins was developed. The system employs two human hepatoma cell lines, Hep 3B and Hep G2, as target cells and agarose affinity electrophoresis with lectins for studies of microheterogeneity of α1‐protease inhibitor (PI), a model glycoprotein synthesized by hepatocytes. As an example for the application of the system, the effect of cytokines on major microheterogeneity of plasma proteins is demonstrated. The results indicate that interleukin 6, transforming growth factor β1and, to some extent, tumor necrosis factor α are directly involved in regulating the pattern of glycosylation of plasma proteinsin vitro, but the major effect is obtained by using combinations of interleukin 6, transforming growth factor β1, tumor necrosis factor a and interleukin 1. In addition, the results underline the dissociation between alteration of gene expression and the changes in the pattern of plasma protein glycosyl

ISSN:0173-0835    

DOI:10.1002/elps.1150101206

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe microheterogeneity profile of human serum transferrin from normal and alcoholic subjects was investigated qualitatively and quantitatively by means of Concanavalin A crossed affinity immunoelectrophoresis and an image analysis program. Differences in amounts of nonreacting transferrin molecules were found, suggesting an increase in triantennary glycosylation of transferrin from alcoholics compared with normal individuals. The increased amount of a highly retarded fraction in crude sera from alcoholics was demonstrated to be artefactual, probably due to entrapment or coprecipitation as the fraction disappeared after repeating the analysis with immunosorbent‐purified transferrin. In conclusion, affinity electrophoresis represents a simple approach for demonstration of variations in the neutral monosaccharides of glycans and can discriminate between transferrin from alcoholics and normal individual

ISSN:0173-0835    

DOI:10.1002/elps.1150101207

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

年代:1989

数据来源: WILEY

摘要:

AbstractIn contrast to the conventional combination of physical, chemical and enzymatic methods used for a structural analysis of glycans in glycoproteins, alternative methods involve affinity electrophoresis as a tool for the detection, characterization, and quantitation of glycoproteins and their carbohydrate moiety, owing to interactions with lectins. Two major approaches involve (i) crossed affino‐immunoelectrophoresis and variations thereof, whereby lectin/glycoprotein interactions occur during the electrophoretic runs, or (ii) affino‐blotting, where the glycoproteins are electrophoretically separated and then immobilized onto a solid support prior to their interaction with lectins. A critical comparison of these two series of techniques is the scope of the present paper. These techniques are of high interest by virtue of their ability at differentiating a classical glycan structure from unusual oligosaccharide side chains. The former structures will usually be qualitatively and quantitatively described with the easy and fast procedures as well as the simple equipment required for crossed affino‐immunoelectrophoresis or affino‐blotting, whereas the latter will be good candidates for further structural a

ISSN:0173-0835    

DOI:10.1002/elps.1150101208

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

年代:1989

数据来源: WILEY

摘要:

AbstractAn affinity electrophoresis system is described to allow determination of dissociation constants of lipopolysaccharide (LPS)‐protein complexes. The LPS ligand is incorporated into polyacrylamide gels by addition to the polyacrylamide‐N, N′‐methyl‐enebisacrylamide polymerization mixture. Quantitative evaluation revealed formation of immobile protein‐ligand complexes. The method was applied both to R‐ and S‐ form LPS fromAcinetobacter calcoaceticus. For a heat‐modifiable outer membrane protein withMr18 000 from strain 69V the dissociation constant was determined to be 0.5 mM (EDTA‐salt extracted R‐LPS) and 0.3 mM (phenol‐chloroform‐petrolether extracted R‐LPS). In comparison, for anotherA. calcoaceticusstrain, CCM 5593, a higher dissociation constant of 1.0 mM (phenol‐chloroform‐petrolether extracted R‐LPS) ‐ indicative of lower affinity ‐ was obtained. When S‐LPS fromA. calcoaceticus69V was incorporated into the affinity gels, a dissociation constant of 0.02 mMwas determined which indicates much stronger interac

ISSN:0173-0835    

DOI:10.1002/elps.1150101209

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

年代:1989

数据来源: WILEY

摘要:

AbstractA natural sulfated polysaccharide (agaropectin), contained in crude agar, can be used as a medium for electrophoretic separation of hemoglobin mutants, constituting a particular class of protein‐ligand interactions. Mutations which either modify the electrostatic charge at the surface of the hemoglobin molecule or not, have been studied according to their putative interaction with the medium. Using conformational specificities of the hemoglobin molecule, we have also demonstrated that isoelectric focusing on a polyacrylamide gel in the absence of heme ligands represents a useful, convenient and rapid procedure for isolating silent Hb variants in their native form, provided that they exibit an abnormal Bohr effec

ISSN:0173-0835    

DOI:10.1002/elps.1150101210

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe development of rocket enzyme activity electrophoresis for the detection and quantification of various proteinases, lipases and pectinases is presented. Rocket enzyme activity electrophoresis is more sensitive than the radial diffusion assay and often enables distinction between qualitatively different enzymes present in the same samples, whereas the radial diffusion assay only provides information on the overall enzyme activity. However, calibration and optimization of the enzyme activity electrophoretic assay have to be performed for each new enzyme‐substrate system to be analyzed. Some of the common pitfalls in the development of new enzyme activity electrophoretic assays are presented. Enzyme activity electrophoresis can be applied in combination with other electrophoretic assays. Particularly the combination of enzyme activity electrophoresis with various immunoelectrophoretic methods can provide detailed information on the enzymes studie

ISSN:0173-0835    

DOI:10.1002/elps.1150101211

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

年代:1989

数据来源: WILEY