Detection and use of recombinant staphylococcal protein A fusion proteins to localize nucleic-acid-binding domains of proteins

Chi V. Dang

Research output: Contribution to journalArticle

Abstract

We developed a rapid method to visualize recombinant staphylococcal protein A fused to segments of another protein and detect DNA-binding proteins simultaneously. Fusion genes were expressed in Escherichia coli to produce fusion proteins of protein A and segments of c-myc oncoprotein. Polypeptides from total E. coli lysates were separated in sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. Without the use of an antigen-specific antibody, protein A/c-myc fusion proteins were visualized by incubation of nitrocellulose membranes with horseradish-peroxidase (HRP)-conjugated immunoglobulin, the Fc region of which directly binds protein A. The sensitivity of detection of the protein A/c-myc fusion proteins was greatly enhanced by incubation of nitrocellulose membranes with rabbit immunoglobulin before incubation with HRP-conjugated goat anti-rabbit antibody. Prior to incubation of nitrocellulose membranes with immunoglobulin, the membranes were incubated with radiolabeled DNA to visualize DNA-binding proteins by autoradiography. Purified staphylococcal protein A does not bind DNA, whereas a segment of c-myc oncoprotein fused to protein A binds DNA in vitro. These methods may be applied to identify nucleic-acid-binding domains of other proteins without prior purification of the fusion proteins.

Original languageEnglish (US)
Pages (from-to)313-317
Number of pages5
JournalAnalytical Biochemistry
Volume174
Issue number1
DOIs
StatePublished - 1988

Fingerprint

Recombinant proteins
Staphylococcal Protein A
Recombinant Proteins
Nucleic Acids
Carrier Proteins
Fusion reactions
Collodion
Membranes
Proteins
Proto-Oncogene Proteins c-myc
Oncogene Proteins
DNA-Binding Proteins
Horseradish Peroxidase
Escherichia coli
Immunoglobulins
DNA
Rabbits
Immunoglobulin Fc Fragments
Antibodies
Gene Fusion

Keywords

  • c-myc
  • DNA-binding
  • fusion genes
  • fusion proteins
  • immunoblotting
  • staphylococcal protein A

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Detection and use of recombinant staphylococcal protein A fusion proteins to localize nucleic-acid-binding domains of proteins. / Dang, Chi V.

In: Analytical Biochemistry, Vol. 174, No. 1, 1988, p. 313-317.

Research output: Contribution to journalArticle

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