UNIT 12.8 Rapid Separation of Protein-Bound DNA from Free DNA Using Nitrocellulose Filters
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Papoulas, O. 2001. Rapid Separation of Protein-Bound DNA from Free DNA Using Nitrocellulose Filters. Current Protocols in Molecular Biology. 36:12.8:12.8.1–12.8.9.
- Published Online: 1 MAY 2001
- Published Print: OCT 1996
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 Alternate Protocol 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 Support Protocol describes how the DNA can be recovered from the filters for further analysis by gel electrophoresis or amplification and cloning.