Purification of DNA‐Binding Proteins Using Biotin/Streptavidin Affinity Systems

Lewis A. Chodosh1

1 Massachusetts Institute of Technology and Harvard Medical School, Cambridge and Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.6
DOI:  10.1002/0471142727.mb1206s36
Online Posting Date:  May, 2001
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Short fragments of DNA‐either natural or formed from oligonucleotides‐containing a high‐affinity site for a DNA‐binding protein provide a powerful tool for purification. The biotin/streptavidin purification system is based on the tight and essentially irreversible complex that biotin forms with streptavidin. In this procedure, a DNA fragment is prepared that contains a high‐affinity binding site for the protein of interest, and a molecule of biotinylated nucleotide is then incorporated into one of the ends of the DNA fragment. The protein of interest binds to the DNA, and then this complex binds (via the biotin moiety) to the tetrameric protein streptavidin. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently isolated by adsorption onto a biotin‐containing resin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin‐containing resin. Proteins remaining in the supernatant are removed by washing, and the resin‐bound protein is then eluted with a high‐salt buffer. An alternate protocol describes a microcolumn method that is useful for larger volumes of biotin‐cellulose resin. This method is also used to elute the protein in as small a volume (i.e., as high a concentration) as possible. Another variation on the basic procedure is provided in which streptavidin‐agarose is employed.

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Table of Contents

  • Alternate Protocol 1: Purification Using a Microcolumn
  • Alternate Protocol 2: Purification Using Streptavidin‐Agarose
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1:

  • Plasmid DNA with desired binding site
  • Appropriate restriction endonucleases (unit 3.1)
  • Biotin‐11‐dUTP ( BRL)
  • Labeled and unlabeled dNTPs (unit 3.4)
  • Klenow fragment of E. coli DNA polymerase I (unit 3.5)
  • TE buffer ( appendix 22)
  • Biotin‐cellulose (Pierce)
  • recipeBiotin‐cellulose binding buffer
  • Bovine serum albumin (BSA)
  • Bulk carrier DNA [e.g., poly(dI‐dC)⋅poly(dI‐dC), salmon sperm DNA, or E. coli DNA]
  • recipeBiotin‐cellulose elution buffer
  • Protein solution
  • Streptavidin (Celltech Ltd.; may be stored as 5 mg/ml stock for at least 2 months)
  • DEAE membrane (Schleicher & Schuell NA45)
  • 0.025‐µm filter discs (Millipore VS)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5), mobility‐shift DNA‐binding assay (unit 12.2), and ethidium bromide dot quantitation (unit 2.6)

Alternate Protocol 1: Purification Using a Microcolumn

  Additional Materials
  • Siliconized glass wool ( appendix 3B)
  • 1.0‐ml pipet tip
  • Ring stand

Alternate Protocol 2: Purification Using Streptavidin‐Agarose

  Additional Materials
  • Streptavidin‐agarose
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Literature Cited

Literature Cited
   Chodosh, L.A., Carthew, R.W., and Sharp, P.A. 1986. A single polypeptide possesses the binding and transcription activities of the adenovirus major late promoter. Mol. Cell. Biol. 6:4723‐4733.
   Grabowski, P.J. and Sharp, P.A. 1986. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science. 233:1294‐1299.
   Haeuptle, M.‐T., Aubert, M.L., Kjiane, J., and Kraehenbuhl, J.‐P. 1983. Binding sites for lactogenic and somatogenic hormones from rabbit mammary gland and liver. J. Biol. Chem. 258:305‐314.
   Kadonaga, J.T. and Tjian, R. 1986. Affinity purification of sequence‐specific DNA binding proteins. Proc. Natl. Acad. Sci. U.S.A. 83:5889‐5893.
   Kasher, M.S., Pintel, D., and Ward, D.C. 1986. Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin‐biotin interactions. Mol. Cell. Biol. 6:3117‐3127.
   Rosenfeld, P.J. and Kelly, T.J. 1986. Purification of nuclear factor I by DNA recognition site affinity chromatography. J. Biol. Chem. 261:1398‐1408.
Key References
   Chodosh et al., 1986. See above.
  Describes the biotin/streptavidin‐agarose microcolumn procedure from which the second alternate protocol of this unit was drawn.
   Kasher et al., 1986. See above.
  Describes a biotin‐cellulose/streptavidin procedure similar to the of this unit.
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