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N H Heegaard

Publications and source records attributed to N H Heegaard.

46 records · Page 3Linked to original sources
PubMed1991

Separation of normal human erythrocyte membrane proteins by high resolution two-dimensional gel electrophoresis.

Different factors influencing two-dimensional gel electrophoresis of red cell membrane proteins were studied: membrane preparation and sample solubilization with a special regard to proteolytic artifacts, urea addition, slab gel acrylamide concentrations and silver staining methods. Spot patterns were analyzed both visually and by means of computer-assisted densitometry. A resolution of around 450 spots was achieved on 10% acrylamide slab gels. The reproducibility of the whole two-dimensional gel electrophoresis procedure was assessed by analysis of computer generated spot densities on gels which were run simultaneously with the same sample. It was shown that the standard red cell membrane preparation method does not lead to proteolysis, contamination by cytosolic proteins, or proteins from other cell types. In comparison with previous studies the relatively high resolution seemed to be due to a high solubilization efficiency combined with the use of a sensitive silver staining method.

Blood Proteins↗
PubMed1990

Immunochemical characterization of interactions between circulating autologous immunoglobulin G and normal human erythrocyte membrane proteins.

Autologous immunoglobulin G present during electrophoresis of human erythrocyte membrane proteins influenced the electrophoretic mobility of some of the proteins. Different types of non-ionic detergents were used for solubilization of the membranes and together with experiments using dimyristoylphosphatidylcholine-derived erythrocyte membrane vesicles this indicated that IgG binds to spectrin, ankyrin, and band 3 protein. The binding was independent on proteolysis and not due to unspecific protein-protein interactions. Immunoblotting experiments also showed binding to polypeptide bands in the spectrin and ankyrin regions and demonstrated the presence of erythrocyte-associated IgG. The reactivity may be due to natural autoantibodies involved in the clearance of cellular debris in vivo. Whether the observations are of relevance for the putative immune-mediated clearance of old erythrocytes from the circulation remains to be established.

ABO Blood-Group System↗
PubMed1990

Visualization of alkaline phosphatase-labelled antibodies on immunoblots by means of formazan staining using indoxyl phosphate and thiazolyl blue.

Alkaline phosphatase-conjugated secondary antibodies on blotting membranes were visualized by means of a modified formazan staining method (Hodson & Skillen 1988). It is based on the standard 5-bromo-4-chloro-3-indolyl phosphate/Nitroblue tetrazolium method, but employs different buffer conditions and thiazolyl blue instead of Nitroblue. It gave a sensitivity of 1 ng albumin in a 12 mm2 dot and less background staining on dot blots than the standard method. The new protocol provides an advantage with regard to ease of staining and preparation of reagents while it equals the former method with respect to sensitivity.

Alkaline Phosphatase↗
PubMed1990

Affinity electrophoresis in agarose gels. Theory and some applications.

Affinity electrophoresis is the electrophoresis of components which interact during the electrophoresis procedure. Among the numerous modifications of the basic principle, affinity electrophoresis in agarose gels combined with subsequent immunochemical detection (crossed affinity immunoelectrophoresis) has emerged as a useful means of characterizing biospecific macromolecular interactions. Demonstration of ligand-binding proteins, enumeration of binding sites, evaluation of microheterogeneous forms, and estimation of binding constants can be achieved with this procedure even when analyzing small amounts of unpurified material. With an emphasis on interactions between lectins and glycoproteins as model systems, the principles and applicability of crossed affinity immunoelectrophoresis are exemplified. This includes examples of modifications developed for screening for lectins in plant extracts, for estimation of the binding capacity of affinity matrices, and for the calculation of dissociation constants.

Antibodies↗
PubMed1989

Has immunoblotting replaced electroimmunoprecipitation? Examples from the analysis of autoantigens and transglutaminase-induced polymers of the human erythrocyte membrane.

The virtues and drawbacks of immunoblotting and electroimmunoprecipitation in the characterization of macromolecules in crude mixtures are presented. Interactions between autoantibodies and human erythrocyte membrane proteins were studied by means of crossed-affinoimmunoelectrophoresis with autologous immunoglobulins incorporated into the first dimension gel and by immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis separated erythrocyte membrane proteins with autologous immunoglobulins as primary antibodies. Substrates for transglutaminase in calcium-activated human erythrocyte membranes were examined by immunoelectrophoretic and immunoblotting methods. The experiments concerning autoantibodies complemented each other and showed that epitopes on Band 3 protein, spectrin and ankyrin are recognized by circulating immunoglobulin autoantibodies in normal individuals. The polymer experiments showed the presence of spectrin, ankyrin, Band 3, Band 4.1, glucose transporter, actin and haemoglobin epitopes in the polymer (Mr 3.10(6)-5.10(6]. It is concluded that the two techniques complement each other. The most evident advantage of immunoblotting is its sensitivity and applicability while electroimmunoprecipitation in some instances allows an easier identification of distinct protein species and still has a role for quantification and certain monitoring purposes.

Autoantibodies↗
PubMed1989

Concanavalin A crossed affinity immunoelectrophoresis and image analysis for semiquantitative evaluation of microheterogeneity profiles of human serum transferrin from alcoholics and normal individuals.

The 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 individuals.

Adult↗
PubMed1989

Hereditary, complete deficiency of complement factor H associated with recurrent meningococcal disease.

Complement factor H (beta-1H globulin) is an important regulatory protein which inhibits the spontaneous complement activation via the alternative pathway. We describe a 15-year-old girl without any detectable factor H in plasma. She has had two episodes of meningococcal disease, but is otherwise completely healthy. Secondary to the factor-H deficiency, the levels of factor B, properdin, C3, and C5-C9 were strongly reduced due to spontaneous in vivo activation of the alternative complement pathway. Plasma C3dg was strongly elevated in spite of the factor-H deficiency; apparently erythrocyte CR1 substitutes for factor H in C3 degradation. Neither C3 nor complement lesions were demonstrable on her erythrocytes which did, however, show increased, spontaneous haemolysis in vitro in citrate plasma, but not in serum. The patient is a single child and her parents, who are unrelated and healthy, had half-normal levels of factor H. This reduction of factor H is sufficient to cause increased, spontaneous activation of the alternative pathway.

Antibodies, Bacterial↗
PubMedDate not supplied

Preparative capillary electrophoresis and mass spectrometry for the identification of a putative heparin-binding site in amyloid P component.

A heparin-binding peptide fragment from chymotrypsin-treated human serum amyloid P component (SAP) was demonstrated by affinity CE. The peptide was found in a fraction of peptides that were not separated well by reversed-phase HPLC. On the basis of mass determination by laser desorption mass spectrometry after preparative CE, the fragment could be placed in the parent protein structure. Thus, in the course of the study of structure-function relationships of SAP, CE was helpful for the examination of peptide fragments from proteolytic digests that were poorly separated by standard reversed-phase HPLC methods and for the purification of peptides in the mixture.

Binding Sites↗