An Artefact in Quantitative Immunoelectrophoresis of Spectrin Caused by Proteolytic Activity in Antibody Preparations

Bjerrum, Ole J.; Ramlau, J.; Clemmesen, I.; Ingild, A.; Bøg-Hansen, T.C.

doi:10.1111/j.1365-3083.1975.tb03812.x

Cited by 63 publications

(21 citation statements)

References 19 publications

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“…Similarly, precipitates 5 and 17 were often difficult to follow. multiple products of proteolytic digestion caused by plasmin (14). Differences were occasionally noted in the quantities of some of the minor immunogens and also in the degree of heterogeneity between different membrane extracts (compare Fig.…”

Section: Materials and Methods Preparation Of Protoplasts And Membranmentioning

confidence: 99%

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Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Owen¹,

Saltón²

1975

Proc. Natl. Acad. Sci. U.S.A.

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on the surface. Of these antigens the major one has been identified as a succinylated mannan. Five of the principal immunoprecipitates unaffected by absorption with protoplasts were shown by zymograms to possess the following enzymic activities: succinate dehydrogenase (EC 1.3.99.1), ATPase (EC 3.6.1.3), NADH dehyrogenase (EC 1.6.99.3)(two separate components), and malate dehydrogenase (EC 1.1.1.37). These enzymes or enzyme-complexes are, therefore, not expressed on the outer surface of the protoplast membrane.Bacterial membranes perform many of the functions found in separate membranous organelles in the eukaryotic cell. Thus functions of electron transport, of active transport, and the biosynthesis of phospholipids and cell walls all occur in the bacterial membrane (1). In membranes of Gram-positive bacteria two principal regions, the plasma or cytoplasmic membrane and the mesosome, are recognizable (2) and these two entities have been isolated as homogeneous fractions from several species (3). Our investigations have been directed towards the elucidation of the structure-function relationships of the membranes of Micrococcus lysodeikticus by a multidisciplinary approach combining biochemical, immunochemical, and electron microscopic techniques. With conventional immunochemical methods, earlier studies in this laboratory showed the presence of three principal immunoprecipitates when membrane fractions of M. lysodeikticus reacted with membrane antiserum (4). These results did not reflect the anticipated complexity of the membrane, considering the multiplicity of the functions it performs and the number (about 30) of individual polypeptides found by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (5, 6). Methods for the preparation of "total" membranes (i.e., plasma and mesosomal membranes), plasma membranes, and purified mannan from M. lysodeikticus are detailed elsewhere (5,8). Triton X-100 extracts of plasma membranes were obtained by treatment with detergent (final concentrations of 4%, w/v) for 1 hr at 220. Membrane residues (about 20%) sedimentable at 17,500 X g for 45 min were removed and the supernatant fractions (19 mg of protein per ml) were retained for analysis.Preparation of Antisera. Rabbits were immunized with "total" membranes (2 mg) given initially into both footpads and intradermally with Freund's complete adjuvant. Further injections of antigen without adjuvant were given weekly by subcutaneous injection at multiple sites. Subsequently, sera from four consecutive bleedings were pooled, and immunoglobulins were partially purified by precipitation with ammonium sulfate and dialysis against acetate buffer, pH 5.0 (9), and then concentrated by ultrafiltration to one-tenth of the original serum volume.Absorption of Anti-Membrane Serum with Protoplasts. Concentrated immunoglobulins (2.0 ml) were dialyzed against Buffer II (0.8 M sucrose, 10 mM Mg2+, 70 mM NaCl, 50 mM Tris'HCl, pH 8.6) and the final volume was adjusted to 6.0 ml. Aliquots (1.0 ml) were incubated for 1 hr at 22°with 0-3....

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Section: Materials and Methods Preparation Of Protoplasts And Membranmentioning

confidence: 99%

“…In control runs, the concanavalin A gel and its spacer gel were replaced with a single spacer gel (15 mm X 82 mm). Electrophoresis in the second dimension was performed at 2.5 V/cm for [12][13][14][15][16][17][18] hr.…”

Section: Materials and Methods Preparation Of Protoplasts And Membranmentioning

confidence: 99%

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Owen¹,

Saltón²

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…Antibodies against an aqueous extract of hair and dander from four different breeds of cow (black and white cattle, red Danish milk breed, Danish Jersey breed and Charoláis), prepared essen tially as for St.C-75, were raised in 3 rabbits and purified according to Harhoe and Ingild [ 15], 10 ml/l of Aprotinin (Novo, Mainz, LRG) was added to avoid proteolytic degradation of the antigens in the second-dimension gel in immunoelectrophoretic analysis [5], The antibodies were stored at 4 °C.…”

Section: Antibodiesmentioning

confidence: 99%

Isolation and Partial Characterisation of Three Major Allergens in an Extract from Cow Hair and Dander

Prahl

Bucher

Plesner

et al. 1982

Int Arch Allergy Immunol

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Three ‘major allergens’ of an extract from cow hair and dander were isolated by means of anion-exchange chromatography and hydrophobic chromatography. Two of the isolated ‘major allergens’ were immunochemically pure and the third contained less than 1% impurities. The molecular weights were 2.0, 2.2 and 2.4 × 10⁴ daltons, and pi was about 4.0. The amino acid content revealed no specific traits. The protein part of the major allergens constituted about 60% (w/w). The allergenic activity of the isolated allergens was verified in RAST-inhibition experiments and in prick tests.

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“…The following decreasing order of inten sity of protease activity was observed: AF (fractions 13-18, maximal at 14); TS (fractions [13][14][15][16][17][18][19]. maximal at 14) and NS (fractions 12 17, maximal at 13).…”

Section: Isolation O F Procoagulant Factormentioning

confidence: 99%

Investigation of the Procoagulant Activity Present in Ascitic Fluid and Serum of Mice Bearing the Landschütz Ascites Carcinoma

Morrice

McIntosh²,

Thomson³

1987

Oncology

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The direct procoagulant activity (PCA) of murine tumour cells was found to be more than three orders of magnitude greater than an equivalent concentration of either resident or Corynehacterium parvum-elicited, exudate peritoneal cells. Similarly, a soluble PCA was detected in the extracellular culture medium of only the tumour cells. Studies on the procoagulant nature of the serum and ascitic fluid of tumour-bearing animals suggested that the ascitic fluid may contain a unique PCA factor(s). This activity could not, however, be resolved from inherent procoagulant factors, either by gel filtration or ammonium sulphate fractionation, and a more specific assay for, say, a single enzymic reaction in the coagulation cascade would be required to identify the tumour-associated activity.

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An Artefact in Quantitative Immunoelectrophoresis of Spectrin Caused by Proteolytic Activity in Antibody Preparations

Cited by 63 publications

References 19 publications

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Isolation and Partial Characterisation of Three Major Allergens in an Extract from Cow Hair and Dander

Investigation of the Procoagulant Activity Present in Ascitic Fluid and Serum of Mice Bearing the Landschütz Ascites Carcinoma

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An Artefact in Quantitative Immunoelectrophoresis of Spectrin Caused by Proteolytic Activity in Antibody Preparations

Cited by 63 publications

References 19 publications

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Isolation and Partial Characterisation of Three Major Allergens in an Extract from Cow Hair and Dander

Investigation of the Procoagulant Activity Present in Ascitic Fluid and Serum of Mice Bearing the Landsch&#252;tz Ascites Carcinoma

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Product

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About

Investigation of the Procoagulant Activity Present in Ascitic Fluid and Serum of Mice Bearing the Landschütz Ascites Carcinoma