Quantitation of proteins by dot-immunobinding assay A comparison of visualization methods using eukaryotic initiation factor 2 and a monospecific antibody

Scot Kimball, Sharon L. Rannels, Mary Beth Elensky, Leonard "Jim" Jefferson

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Various visualization methods were compared for quantitation of proteins by the dot-immunobinding assay. Comparisons were carried out using a multi-subunit protein, eukaryotic initiation factor 2, and monospecific antibodies directed against two of the factor's subunits. The protein was spotted onto nitrocellulose and the membranes were incubated with primary antibody. The antigen-antibody complex was visualized by one of six methods using either alkaline phosphatase-, horseradish peroxidase-, or glucose oxidase-conjugated IgG, or colloidal gold-labelled IgG, colloidal gold-labelled IgG with silver enhancement, or 125I-labelled protein A. The amount of secondary antibody bound was quantitated by densitometric scanning of the nitrocellulose membrane after staining or autoradiography. The sensitivity of each of the methods was similar; each of the visualization methods could detect less than 1 ng of protein by the dot-immunobinding assay. Curves of protein concentration vs. densitometric absorbance were found to fit a parabolic relationship (r2 = 0.99) over a wide range of concentrations.

Original languageEnglish (US)
Pages (from-to)217-223
Number of pages7
JournalJournal of Immunological Methods
Volume106
Issue number2
DOIs
StatePublished - Feb 10 1988

Fingerprint

Eukaryotic Initiation Factor-2
Gold Colloid
Collodion
Antibodies
Immunoglobulin G
Proteins
Glucose Oxidase
Membranes
Protein Subunits
Staphylococcal Protein A
Horseradish Peroxidase
Antigen-Antibody Complex
Autoradiography
Silver
Alkaline Phosphatase
Staining and Labeling

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Immunology

Cite this

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abstract = "Various visualization methods were compared for quantitation of proteins by the dot-immunobinding assay. Comparisons were carried out using a multi-subunit protein, eukaryotic initiation factor 2, and monospecific antibodies directed against two of the factor's subunits. The protein was spotted onto nitrocellulose and the membranes were incubated with primary antibody. The antigen-antibody complex was visualized by one of six methods using either alkaline phosphatase-, horseradish peroxidase-, or glucose oxidase-conjugated IgG, or colloidal gold-labelled IgG, colloidal gold-labelled IgG with silver enhancement, or 125I-labelled protein A. The amount of secondary antibody bound was quantitated by densitometric scanning of the nitrocellulose membrane after staining or autoradiography. The sensitivity of each of the methods was similar; each of the visualization methods could detect less than 1 ng of protein by the dot-immunobinding assay. Curves of protein concentration vs. densitometric absorbance were found to fit a parabolic relationship (r2 = 0.99) over a wide range of concentrations.",
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Quantitation of proteins by dot-immunobinding assay A comparison of visualization methods using eukaryotic initiation factor 2 and a monospecific antibody. / Kimball, Scot; Rannels, Sharon L.; Elensky, Mary Beth; Jefferson, Leonard "Jim".

In: Journal of Immunological Methods, Vol. 106, No. 2, 10.02.1988, p. 217-223.

Research output: Contribution to journalArticle

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