A colorimetric assay specific for γ-carboxyglutamic acid-containing proteins

Its utility in protein purification procedures

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17 Citations (Scopus)

Abstract

A colorimetric method for the detection of γ-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without γ-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by γ-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the γ-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second γ-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 μm for the γ-carboxyglutamic acid-containing protein bone Gla protein and up to 27 μm for the amino acid. The detection limit is down to 1 μg of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for γ-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.

Original languageEnglish (US)
Pages (from-to)273-279
Number of pages7
JournalAnalytical Biochemistry
Volume186
Issue number2
DOIs
StatePublished - May 1 1990

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Purification
Assays
Acids
Proteins
Color
Osteocalcin
Membranes
Staining and Labeling
Prothrombin
Complex Mixtures
Screening
Bone
Limit of Detection
Western Blotting
Amino Acids
Bone and Bones
Monitoring

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "A colorimetric assay specific for γ-carboxyglutamic acid-containing proteins: Its utility in protein purification procedures",
abstract = "A colorimetric method for the detection of γ-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without γ-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by γ-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the γ-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second γ-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 μm for the γ-carboxyglutamic acid-containing protein bone Gla protein and up to 27 μm for the amino acid. The detection limit is down to 1 μg of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for γ-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.",
author = "Satoru Nishimoto",
year = "1990",
month = "5",
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doi = "10.1016/0003-2697(90)90079-O",
language = "English (US)",
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pages = "273--279",
journal = "Analytical Biochemistry",
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N2 - A colorimetric method for the detection of γ-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without γ-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by γ-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the γ-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second γ-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 μm for the γ-carboxyglutamic acid-containing protein bone Gla protein and up to 27 μm for the amino acid. The detection limit is down to 1 μg of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for γ-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.

AB - A colorimetric method for the detection of γ-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without γ-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by γ-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the γ-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second γ-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 μm for the γ-carboxyglutamic acid-containing protein bone Gla protein and up to 27 μm for the amino acid. The detection limit is down to 1 μg of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for γ-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.

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