Assessing acetylation of NF-kappaB

Methods. 2005 Aug;36(4):368-75. doi: 10.1016/j.ymeth.2005.03.011.


To achieve its full biological activity, NF-kappaB must undergo a variety of post-translational modifications, including acetylation. Acetylation plays a prominent role in regulating the nuclear action of NF-kappaB. The RelA subunit of NF-kappaB forms the major target of acetylation at several different sites. Acetylation of discrete lysine residues in RelA modulates distinct functions of NF-kappaB, including transcriptional activation, DNA binding, and assembly with its inhibitor IkappaBalpha. Here, we describe the experimental methods that have allowed the detection and functional analysis of acetylated forms of NF-kappaB. Acetylation of NF-kappaB can be studied both in vivo and in vitro. In vivo [3H]acetate labeling assays provides a useful, albeit rather insensitive, method for initial verification of acetylation of either over-expressed or endogenous subunits of NF-kappaB. A second valuable in vivo approach involves the use of anti-acetylated lysine antibodies for immunoblotting. However, the success of this approach varies with the specific antibody employed and the target protein studied. In vitro acetylation assays provide a rapid and sensitive method to validate the involvement of candidate histone acetyltransferases and to map the sites of acetylation. Anti-RelA antibodies that selectively react with site-specific acetylated forms of RelA are a singularly powerful tool for the study of NF-kappaB acetylation both in vivo and in vitro.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetates / immunology
  • Acetates / metabolism
  • Acetylation
  • Acetyltransferases / immunology
  • Acetyltransferases / metabolism
  • Animals
  • Antibodies / immunology
  • Cell Cycle Proteins / immunology
  • Cell Cycle Proteins / metabolism
  • Cell Extracts / isolation & purification
  • Cell Line
  • Clinical Laboratory Techniques*
  • Histone Acetyltransferases
  • Humans
  • Immunoprecipitation / methods
  • Lysine / immunology
  • Lysine / metabolism
  • NF-kappa B / metabolism*
  • Protein Processing, Post-Translational*
  • Radioisotopes / chemistry
  • Transcription Factor RelA
  • Transcription Factors / immunology
  • Transcription Factors / metabolism
  • Transfection / methods
  • p300-CBP Transcription Factors


  • Acetates
  • Antibodies
  • Cell Cycle Proteins
  • Cell Extracts
  • NF-kappa B
  • Radioisotopes
  • Transcription Factor RelA
  • Transcription Factors
  • Acetyltransferases
  • Histone Acetyltransferases
  • p300-CBP Transcription Factors
  • p300-CBP-associated factor
  • Lysine