Demonstration of binding of triton X‐100 to amphiphilic proteins in crossed immunoelectrophoresis

Bjerrum, Ole J.; Bhakdi, Sucharit

doi:10.1016/0014-5793(77)80949-6

Cited by 20 publications

(2 citation statements)

References 13 publications

(5 reference statements)

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“…The antigens were probably of cytoplasmic origin because similar results were obtained with and without saponin in the gels, although this detergent is not always a reliable indicator of a non-membrane origin of proteins (Bjerrum & Bhakdi, 1977), but hydrophobic interaction chromatography appeared to confirm the cytoplasmic origin of the antigens since all the arcs detected were in the cytoplasmic fraction of the eluant. However, some arcs were not detected by Laurell IEP following this technique and some antigens may have remained firmly bound to the column.…”

Section: Discussionmentioning

confidence: 67%

Immunochemical studies on pathotypes of the potato cyst nematodes, Globodera rostochiensis and G. pallida

Fox

Atkinson

1985

Parasitology

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The potential of antigenic differences for discriminating pathotypes of the potato cyst nematodes Globodera pallida and G. rostochiensis has been examined by the use of an antiserum raised to a homogenate of potato cyst nematode larvae. Species-specific antigens were detected among reference pathotypes but more variability was detected among field populations, and cluster analysis was used to interpret the precipitation are pattern produced by Laurell crossed-immunoelectrophoresis. A division into species was seen with this analysis but no definite pathotype groupings were detected. Cross-reaction with other cyst-nematode species was limited to general non-specific precipitation. The antigens were all proteinaceous, did not arise from micro-organisms within the cyst and were mainly hydrophilic with an acidic isoelectric point. Peptidase and acid phosphatase activity was detected in some precipitation arcs but this was not species specific.

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Section: Discussionmentioning

confidence: 67%

Immunochemical studies on pathotypes of the potato cyst nematodes, Globodera rostochiensis and G. pallida

Fox

Atkinson

1985

Parasitology

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“…The analysis performed on human serum protein shows that the plates must be washed with water before a safe identification can be performed, even though the Sudan Blackstained precipitates are more distinct on the wet plates. The method is less sensitive than charge-shift crossed immunoelectrophoresis, but gives results similar to the use of radioactive detergent [6]. The sensitivity is about the same, i. e., only major precipitates can be identified, but the Sudan Black method is much faster, since the results can be directly read from the washed immunoplate.…”

Section: Discussionmentioning

confidence: 97%

Electroimmunochemical analysis of amphiphilic proteins and glycolipids stained with Sudan Black‐containing detergent micelles

Bjerrum¹,

Gerlach²,

Bøg-Hansen³

et al. 1982

Electrophoresis

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Micelles of non-ionic detergents can be colored through incorporation of the hydrophobic dye Sudan Black. This property can be used to identify amphiphilic antigens in complex mixtures. Thus, crossed immunoelectrophoresis with 0.05 % v/v Triton X-100 plus 0.002 % w/v Sudan Black shows selective staining of major amphiphilic proteins from human erythrocytes, thrombocytes and serum, from bovine milk fat globules and from yeast plasma membranes. The method may also give information about glycolipid antigens in detergent-solubilized membrane material, e. g. mannosyl-diinositolphosphoryl-ceramide from yeast. In this case, however, the membrane has to be solubilized with detergent plus Sudan Black and analyzed in detergent-free gels, where a stable micellar structure seems to exist. In a surplus of detergent, the glycolipid antigen may enter new micelles during electrophoresis, reducing the number of epitopes per micelle below two, thus avoiding precipitation. Analysis of purified lipopolysaccharide from Bordetellapertussis followed this pattern, whereas lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli, although heavily influenced by the presence of Triton X-100, were precipitated in the presence of a surplus of detergent. The described method is simple, fast and easy to perform, It requires neither extra equipment nor expensive chemicals. Sometimes heavily stained, detergent-containing precipitates of a non immunological nature may appear, but these can easily be discerned from immunoprecipitates.

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Electrophoretic migration velocity of amphiphilic proteins increases with decreasing Triton X‐100 concentration: A new characteristic for their identification

Børsum¹,

Bjerrum²

1986

Electrophoresis

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The relation between the electrophoretic migration velocity and the concentration of the non‐ionic detergent Triton X‐100 in the buffer was studied by crossed immunoelectrophoresis of hydrophilic and amphiphilic proteins. The migration velocity of hydrophilic human plasma proteins was not affected by the presence of Triton X‐100 in the agarose gels in the concentration range of 1–0 % v/v. In contrast, amphiphilic proteins, like the human erythrocyte proteins glycophorin, band 3 protein and acetylcholinesterase, and the plasma high density lipoprotein (HDL) apolipoprotein, showed increasing migration velocity with decreasing detergent concentration in the gels. An increase of 20–80 % was observed for different amphiphilic proteins when the Triton X‐100 concentration was lowered from 1 % to 0.03 % v/v. This probably reflects the influence of the size of the bound detergent micelle on the charge density of the protein. Thus, by performing corssed immunoelectrophoresis with different concentrations of Triton X‐100 in the first‐dimensional gel it is possible to identify amphiphilic proteins. Presence of 1 % v/v Triton X‐100 in the second‐dimensional gel ensures reliable identification of the precipitates.

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Demonstration of binding of triton X‐100 to amphiphilic proteins in crossed immunoelectrophoresis

Cited by 20 publications

References 13 publications

Immunochemical studies on pathotypes of the potato cyst nematodes, Globodera rostochiensis and G. pallida

Immunochemical studies on pathotypes of the potato cyst nematodes, Globodera rostochiensis and G. pallida

Electroimmunochemical analysis of amphiphilic proteins and glycolipids stained with Sudan Black‐containing detergent micelles

Electrophoretic migration velocity of amphiphilic proteins increases with decreasing Triton X‐100 concentration: A new characteristic for their identification

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