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Description and application of an immunological detection system for analyzing glycoproteins on blots

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Abstract

By introducing the steroid hapten digoxigenin specifically into sugars, a sensitive detection system for glycoproteins on blots has been developed. Sugars are oxidized to obtain aldehyde groups, which then react with digoxigenin-succinyl-ε-amido caproic acid hydrazide. A high-affinity antibody, conjugated to alkaline phosphatase, is used for the detection of the incorporated digoxigenin.

This system allows the detection of nanogram-amounts of glycoproteins on blots, and it's specificity allows a clear distinction of a glycoprotein from a non-glycoprotein. In combination with endo- and exoglycosidases it is very useful for determining the type of carbohydrate linkage in a glycoprotein, and by varying the oxidation conditions, specific labeling of sialic acids and terminal galactoses can be achieved.

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Abbreviations

CpY:

Carboxypeptidase Y

CHO:

Chinese hamster ovary

Dig-hydrazide:

digoxigenin-succinyl-ε-caproic acid hydrazide

〈Dig〉Fab-AP:

polyclonal sheep anti-digoxigenin Fab fragments, conjugated with alkaline phosphatase

NBT:

nitroblue tetrazolium chloride

SDS-PAGE:

sodium dodecylsulfate-polyacrylamide gel electrophoresis

TCA:

trichloroacetic acid

X-phosphate:

5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt

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Authors and Affiliations

  1. Biochemica Research Center, Boehringer Mannheim GmbH, D-8132, Tutzing/Obb., Federal Republic of Germany

    Anton Haselbeck & Wolfgang Hösel

Authors
  1. Anton Haselbeck
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  2. Wolfgang Hösel
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Haselbeck, A., Hösel, W. Description and application of an immunological detection system for analyzing glycoproteins on blots. Glycoconjugate J 7, 63–74 (1990). https://doi.org/10.1007/BF01050403

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  • DOI: https://doi.org/10.1007/BF01050403

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