Identification of Novel DNA‐Binding Proteins Using DNA‐Affinity Chromatography/Pull Down

Brandon L. Jutras1, Ashutosh Verma1, Brian Stevenson1

1 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1F.1
DOI:  10.1002/9780471729259.mc01f01s24
Online Posting Date:  February, 2012
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Abstract

This units presents methods through which one may isolate and identify novel bacterial DNA‐binding proteins. Briefly, the DNA sequence of interest is affixed to beads, and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low‐salt concentrations will remove non‐adhering and low‐specificity DNA‐binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA‐binding proteins. Eluted proteins may then be identified by standard proteomic techniques. Curr. Protoc. Microbiol. 24:1F.1.1‐1F.1.13. © 2012 by John Wiley & Sons, Inc.

Keywords: DNA‐binding protein; prokaryote; gene regulation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cell Lysis by Sonication with Freeze Thaw
  • Alternate Protocol 1: Cell Lysis Using BPER‐II Extraction
  • Alternate Protocol 2: Cell Lysis by Vigorous Vortexing and Freeze Thaw Cycles
  • Basic Protocol 2: DNA‐Affinity Chromatography: Borrelia burgdorferi and Leptospira interrogans
  • Basic Protocol 3: Visualization and Identification of DNA‐Binding Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cell Lysis by Sonication with Freeze Thaw

  Materials
  • Bacterial culture of interest
  • Ultrapure or nuclease‐free water
  • Ice
  • BS/THES buffer (see recipe)
  • −80°C freezer
  • Sonicator for disrupting bacterial cells

Alternate Protocol 1: Cell Lysis Using BPER‐II Extraction

  Materials
  • Frozen cell pellets (see protocol 1)
  • BPER‐II (Thermo Scientific)
  • BS/THES buffer (see recipe)
  • Lysozyme
  • Vortex
  • 0.2‐µm filter
  • Shaking incubator
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge

Alternate Protocol 2: Cell Lysis by Vigorous Vortexing and Freeze Thaw Cycles

  Materials
  • Cell suspension (see protocol 1, step 5)
  • BS/THES buffer (see recipe)
  • Ice
  • −80°C freezer
  • Vortex mixer
  • Centrifuge

Basic Protocol 2: DNA‐Affinity Chromatography: Borrelia burgdorferi and Leptospira interrogans

  Materials
  • M‐280 Dynabeads (Invitrogen)
  • 2× B/W buffer (see recipe)
  • Biotinylated DNA probe (IDT DNA Technologies)
  • Nuclease‐free water
  • TE buffer [1 M Tris⋅Cl, pH 8.0 ( appendix 2A), and 0.5 M EDTA, pH 8.0 ( appendix 2A)]
  • BS/THES buffer (see recipe)
  • Nonspecific inhibitor DNA: e.g., poly dI‐dC or dA‐dT
  • Elution buffer with varying NaCl concentrations (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Magnetic column: PolyATtract System 1000 (Promega)
  • 200‐µl pipets
  • Rocker/belly dancer (e.g., Labnet Revolver)
  • 70°C heat block
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Figures

Videos

Literature Cited

Literature Cited
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