![]() The protein‑DNA complex is treated lightly with DNase I, so that on average each DNA molecule is cleaved once. DNase footprint analysis: DNase I will cut at many (but not all) phosphodiester bonds in the free DNA. One can use a combination of a 3' to 5' exonuclease (ExoIII) and a 5' to 3' exonuclease (l exonuclease) to map both edges. Exonuclease protection assay: The protein will block the progress of an exonuclease, so the protected fragment extends from the labeled site to the edge of the protein furtherest from the labeled site. An end‑labeled DNA fragment in complex with protein is treated with a nuclease (or other cleaving reagent), and the protected fragments are resolved on a denaturing polyacrylamide gel, and their sizes measured. to an adjacent sequence that is distorted from normal B‑form). The presence of a protein will either protect a segment of DNA from attack by a nuclease or other degradative reagent, or in some cases will enhance cleavage (e.g. To what sequence in the probe DNA is the protein binding? ![]() Nitrocellulose binding: Free duplex DNA will not stick to a nitrocellulose membrane, but a protein‑DNA complex will bind.Ģ. The proteins forming complexes A and B do not recognize an Oct1-binding site (lanes 12-14).ī. Hence the protein could be Sp1 or a relative of it. It recognizes an Sp1-binding site, as shown by the ability of an oligonucloetide with an Sp1-binding to compete for complex A, but not complex B (lanes 9-11). This experiment also provides some information about the identity of the protein forming complex A. coli DNA does not compete effectively (compare lanes 6-7 with lanes 3-5). An excess of unlabeled oligonucleotide with the same sequence as the labeled probe (“self”) prevents formation of the complexes with labeled probe, whereas “nonspecific DNA” in the form of E. ![]() This is shown by the competition assays in lanes 3-8. The proteins in complexes A and B recognize specificDNA sequences in the probe. In this example, two proteins recognize sequences in the labeled probe, forming complexes A and B (lane 2). Diagram of results from an electrophoretic mobility shift assay By incubating the probe and proteins in the presence of increasing amounts of competitor DNA fragments, one can test for specificity and even glean some information about the identity of the binding protein.įigure 3.2.4. The presence of a slowly moving signal is indicative of a complex between the DNA probe and some protein(s). If the DNA fragment binds to the protein, the complex will migrate much slower in the gel than does the free probe it moves with roughly the mobility of the bound protein. A selected restriction fragment or synthetic duplex oligonucleotide is labeled (to make a probe) and mixed with a protein (or crude mixture of proteins). Many protein‑DNA complexes are sufficiently stable that they will remain together during electrophoresis through a (nondenaturing) polyacrylamide gel. Electrophoretic mobility shift assay (EMSA), or gel retardation assay: This assay will test for the ability of a particular sequence to form a complex with a protein. Does a protein bind to a particular region?Ī.
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