Immunofluorescence Analysis Using Epitope-Tagged Proteins

In Vitro System
  • Amjad Javed
  • Sayyed K. Zaidi
  • Soraya E. Gutierrez
  • Christopher J. Lengner
  • Kimberly S. Harrington
  • Hayk Hovhannisyan
  • Brian C. Cho
  • Jitesh Pratap
  • Shirwin M. Pockwinse
  • Martin Montecino
  • André J. van Wijnen
  • Jane B. Lian
  • Janet L. Stein
  • Gary S. Stein
Part of the Methods in Molecular Biology™ book series (MIMB, volume 285)

Abstract

The resolution that can be attained in assessment of the intranuclear localization of cellular proteins is dependent on specificity of the antibodies. Primary antibodies should be well characterized. We recommend testing antibody specificity by immunoblotting. In general, monoclonal antibodies have a selective advantage of recognizing only a single epitope but may not produce a high titer. In contrast, polyclonal antibodies generally have a high titer but may recognize multiple epitopes that may result in crossreactivity with several proteins because of similar amino acid sequences. The problem of low titer antibody and poor antigenicity can be overcome by marking the proteins with an epitope “tag.” The most widely used tags in prokaryotic and eukaryotic cells include glutathione S-transferase , β-galactosidase, 6× histidine, hemagglutinin, Xpress, Flag, Myc, and a variety of fluorescent proteins (green fluorescent protein [GFP], cyan fluorescent protein, yellow fluorescent protein, and red fluorescent protein). Caution must be exercised when tagging large proteins (e.g., GFP or β-galactosidase) at the amino or carboxy termini to avoid interference with folding that can influence activity. Most “epitope tags” are very short sequences, thereby having little or no crossreactivity with other cellular proteins. The well-characterized antibodies that are commercially available against these tags are also conjugated to fluorescent molecules. These conjugated reagents reduce both the time and assessment of chemical needed to visualize the final product.

Keywords

Phenol Codon DMSO Glutathione HEPES 

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Amjad Javed
    • 1
  • Sayyed K. Zaidi
    • 1
  • Soraya E. Gutierrez
    • 1
  • Christopher J. Lengner
    • 1
  • Kimberly S. Harrington
    • 1
  • Hayk Hovhannisyan
    • 1
  • Brian C. Cho
    • 1
  • Jitesh Pratap
    • 1
  • Shirwin M. Pockwinse
    • 1
  • Martin Montecino
    • 1
  • André J. van Wijnen
    • 1
  • Jane B. Lian
    • 1
  • Janet L. Stein
    • 1
  • Gary S. Stein
    • 1
  1. 1.Department of Cell BiologyUniversity of Massachusetts Medical SchoolWorcesterMA

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