High-Throughput Analysis of the Cell and DNA Site-Specific Binding of Native NF-κB Dimers Using Nuclear Extract Protein-Binding Microarrays (NextPBMs)

Methods Mol Biol. 2021:2366:43-66. doi: 10.1007/978-1-0716-1669-7_4.

Abstract

Nuclear factor-kappa B (NF-κB) transcription factors coordinate gene expression in response to a broad array of cellular signals. In vertebrates, there are five NF-κB proteins (c-Rel, RelA/p65, RelB, p50, and p52) that can form various dimeric combinations exhibiting both common and dimer-specific DNA-binding specificity. In this chapter, we describe the use of the nuclear extract protein-binding microarray (nextPBM), a high-throughput method to characterize the DNA binding of transcription factors present in cell nuclear extracts. NextPBMs allow for sensitive analysis of the DNA binding of NF-κB dimers and their interactions with cell-specific cofactors.

Keywords: DNA binding; NF-kB; Nuclear extract; Protein-binding microarrays.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • DNA / genetics
  • DNA / metabolism
  • NF-kappa B / metabolism
  • NF-kappa B p50 Subunit / genetics
  • NF-kappa B p50 Subunit / metabolism
  • Plant Extracts
  • Protein Array Analysis*
  • Protein Binding
  • Signal Transduction
  • Transcription Factor RelA / genetics
  • Transcription Factor RelA / metabolism

Substances

  • NF-kappa B
  • NF-kappa B p50 Subunit
  • Plant Extracts
  • Transcription Factor RelA
  • DNA