Measuring Lymphocyte Transcription Factor Activity by ELISA

Jesica McCue1, Brian Freed1

1 University of Colorado Health Sciences Center, Denver, Colorado
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.5
DOI:  10.1002/0471140856.tx1805s22
Online Posting Date:  January, 2005
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Abstract

Adequate immune function requires the complex interaction between cells via a series of biochemical and molecular events, culminating in altered gene expression. Initiation of transcription by sequence‐specific DNA‐binding proteins known as transcription factors (TFs) is the principle point at which the expression of most genes is regulated. Thus, an understanding of nuclear events affected by environmental toxicants and their mechanisms of actions is critical to understanding toxic phenomena.

Colorimetric enzyme‐linked immunosorbent assay (ELISA)‐based procedures have been developed to detect specific transcription factor DNA‐binding activity in cell extracts. Up to 96 reactions can be performed in 3 to 4 hr. Extracts are added to the 96‐well plate precoated with a transcription factor DNA‐binding consensus sequence and detected with an antibody specific to the transcription factor of interest. In short, ELISA provides increased speed and throughput, and allows improved sensitivity and convenience over the traditional methods.

Keywords: Transcription Factor; DNA‐binding Activity; ELISA; Transcriptional Regulation

     
 
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Table of Contents

  • Basic Protocol 1: DNA‐Binding ELISA
  • Support Protocol 1: Coat Plates with DNA
  • Support Protocol 2: Prepare Nuclear Extracts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: DNA‐Binding ELISA

  Materials
  • Blocking buffer (see recipe)
  • ELISA plate precoated with wild‐type and mutant dsDNA (see protocol 2)
  • Nuclear extract (see protocol 3)
  • PBS (see recipe)
  • Wild‐type competitor oligonucleotides
  • Wash buffer (see recipe)
  • Primary antibody
  • Secondary antibody e.g., anti‐rabbit IgG‐HRP or anti‐mouse IgG‐HRP (Clontech)
  • TMB substrate (Sigma)
  • Stop solution: 1 M H 2SO 4 (optional)
  • Microtiter plate reader

Support Protocol 1: Coat Plates with DNA

  Materials
  • ssDNA: consensus sequence of transcription factor of interest (e.g., BD Mercury TransFactor Kit, BD Biosciences)
  • TE buffer (see recipe)
  • Reacti‐Bind DNA coating solution (Pierce)
  • PBS (see recipe)
  • ELISA plate precoated with wild‐type and mutant dsDNA
  • 13 × 100–mm glass test tube

Support Protocol 2: Prepare Nuclear Extracts

  Materials
  • Cells, e.g., PBMC (>106 cells)
  • PBS (see recipe), ice cold
  • Hypotonic buffer (see recipe), ice cold
  • High‐salt buffer (see recipe), ice cold
  • Low‐salt buffer (see recipe), ice cold
  • BCA or Bradford protein concentration kit (Sigma)
  • Liquid nitrogen
  • 1.5‐ml microcentrifuge tubes, prechilled
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Figures

  •   FigureFigure 18.5.1 Inhibition of p50 DNA binding by cigarette smoke aldehydes. The ability of the various aldehydes to inhibit the interaction between recombinant p50 and the NF‐κB promoter region was measured using a BD Mercury NF‐κB/p50 TransFactor Kit. A saturating concentration of p50 (160 pmol) was treated with 0 to 100 mM acrolein (empty circles), crotonaldehyde (empty squares), acetaldehyde (colored squares), propionaldehyde (colored circles), or butyraldehyde (colored diamonds) and then added to wells coated with NF‐κB consensus sequence (GGGGATCCC). The amount of p50 bound was measured using anti‐p50, HRP‐conjugated anti‐rabbit IgG, and TMB and the results expressed as mOD/min. Results are the mean ±SE of two experiments.
  •   FigureFigure 18.5.2 Acrolein blocks induction of p50 DNA activity. Jurkat T cells were treated with 10 µM acrolein (AC) for 3 hr and then stimulated with anti‐CD3 plus PMA for 24 hr. Culture supernatants were analyzed for IL‐2 levels, shown in white numbers as pg/ml. Nuclear extracts were prepared and assayed for p50 DNA binding using a 42‐mer NF‐κB consensus target sequence. NS, non‐stimulated.

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Literature Cited

Literature Cited
   Benotmane, A.M., Hoylaerts, M.F., Collen, D., and Belayew, A. 1997. Nonisotopic quantitative analysis of protein‐DNA interactions at equilibrium. Anal. Biochem. 250:181‐185.
   Brivanlou, A.H. and Darnell, J.E. 2002. Signal transduction and the control of gene expression. Science 295:813‐818.
   Dignam, J.D., Lebovitz, R.M., and Roeder, R.G. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucl. Acids Res. 11:1475‐1489.
   Li‐Weber, M. and Krammer, P.H. 2003. Regulation of IL4 gene expression by T cells and therapeutic perspectives. Natu. Immun. Rev. 3:534‐543.
   Shen, Z., Peedikayil, J., Olson, G.K., Siebert, P.D., and Fang, Y. 2002. Multiple transcription factor profiling by enzyme‐linked immunoassay. BioTechniques 32:1168‐1177.
   Yusuf, I. and Fruman, D.A. 2003. Regulation of quiescence in lymphocytes. Trends Immunol. 24:380‐386.
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