Rapid Separation of Protein‐Bound DNA from Free DNA Using Nitrocellulose Filters

Ophelia Papoulas1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.8
DOI:  10.1002/0471142727.mb1208s36
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Nitrocellulose binds proteins but not double‐stranded DNA. Use of radioactively labeled double‐stranded DNA fragments allows quantitation of DNA bound to the protein, permitting kinetic and equilibrium studies of DNA‐binding interactions. In the basic procedure, purified protein is mixed with double‐stranded DNA and then the mixture is filtered through nitrocellulose, allowing unbound DNA to pass through the filter while the protein (and any DNA interacting with it) is retained. When the binding site of a protein is unknown, the pure protein can be added to a mixture of fragments to select those fragments of DNA for which it has the greatest affinity. Specificity of binding can be influenced by the buffer conditions and filtering regimen. An is provided that creates conditions that disrupt weaker, presumably nonspecific binding interactions, while retaining the stronger binding interactions. The goal is to recover enough of a single input fragment to visualize by subsequent autoradiography. In some cases the quantitation (by scintillation counting) of DNA retained is not sufficient information. A describes how the DNA can be recovered from the filters for further analysis by gel electrophoresis or amplification and cloning.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Alternate Protocol 1: Detection of Specificity in DNA Binding
  • Support Protocol 1: Elution of Bound DNA
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • recipe2× DNA‐binding buffer
  • Double‐stranded, 32P‐labeled DNA
  • Protein sample
  • Bovine serum albumin (BSA; optional)
  • 0.45‐µm nitrocellulose filter discs (13‐, 24‐, or 25‐mm diameter to fit filtration apparatus being used)
  • Flat, smooth‐tip forceps for handling nitrocellulose
  • Filtration apparatus and vacuum source

Alternate Protocol 1: Detection of Specificity in DNA Binding

  Additional Materials
  • recipeFilter elution buffer
  • Carrier nucleic acid
  • Ethanol
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Barkley, M.D., Riggs, A.D., Jobe, A., and Bourgeois, S. 1975. Interaction of effecting ligands with lac repressor and repressor‐operator complex. Biochemistry 14:1700‐1712.
   Fried, M. and Crothers, D.M. 1981. Equilibria and kinetics of lac repressor‐operator interactions by polyacrylamide gel electrophoresis. Nucl. Acids Res. 9:6505‐6525.
   Hinkle, D.C. and Chamberlin, M.J. 1972a. Studies of the binding of Escherichia coli RNA polymerase to DNA I. The role of sigma subunit in site selection. J. Mol. Biol. 70:157‐185.
   Hinkle, D.C. and Chamberlin, M.J. 1972b. Studies of the binding of Escherichia coli RNA polymerase to DNA II. The kinetics of the binding reaction. J. Mol. Biol. 70:187‐195.
   Hinkle, D.C. and Chamberlin, M.J. 1972c. Studies of the binding of Escherichia coli RNA polymerase to DNA III. Tight binding of RNA polymerase holoenzyme to single‐strand breaks in T7 DNA. J. Mol. Biol. 70:197‐207.
   Jones, O.W. and Berg, P. 1966. Studies on the binding of RNA polymerase to polynucleotides. J. Mol. Biol. 22:199‐209.
   Lin, S.‐Y. and Riggs, A.D. 1972. Lac repressor binding to non‐operator DNA: Detailed studies and a comparison of equilibrium and rate competition methods. J. Mol. Biol. 72:671‐690.
   Lin, S.‐Y. and Riggs, A.D. 1975. The general affinity of lac repressor for E. coli DNA: Implications for gene regulation in procaryotes and eucaryotes. Cell 4:107‐111.
   Nirenberg, M. and Leder, P. 1964. RNA codewords and protein synthesis: The effect of trinucleotides upon the binding of sRNA to ribosomes. Science 145:1399‐1407.
   Ptashne, M. 1987. A Genetic Switch: Gene Control and Phage λ, pp.80‐83 and 109‐118. Cell Press, Cambridge, MA and Blackwell Scientific, Boston, MA.
   Riggs, A.D., Suzuki, H., and Bourgeois, S. 1970. Lac repressor‐operator interactions: I. Equilibrium studies. J. Mol. Biol. 48:67‐83.
   Strauss, H.S., Burgess, R.R., and Record, M.T. Jr. 1980a. Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter‐containing fragment: Selectivity exists over a wide range of solution conditions. Biochemistry 19:3496‐3504.
   Strauss, H.S., Burgess, R.R., and Record, M.T. Jr. 1980b. Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter‐containing fragment: Evaluation of promoter binding constants as a function of solution conditions. Biochemistry 19:3504‐3515.
   Strauss, H.S., Boston, R.S., Record, M.T. Jr., and Burgess, R.R. 1981. Variables affecting the selectivity and efficiency of retention of DNA fragments by E. coli RNA polymerase in the nitrocellulose‐filter‐binding assay. Gene 13:75‐87.
Key References
   Ptashne, 1987. See above.
  A straightforward introduction to DNA‐binding interactions and equations and a good place to start for theory.
   Riggs et al., 1970. See above.
  An early paper covering the underlying theory and limitations of the filter‐binding technique. In addition to actual data it provides detail on most applications described in this protocol, and is an excellent source of practical information.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library