lecture13 - Which nt Residues are Contacted by the Protein?...

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Which nt Residues are Contacted by the Protein? Modification Interference Method --tests ability of partially modified DNA to bind protein --DNA for binding analysis is partially modified such that approx. every molecule has one modification --residues that are not critical to protein binding will be modified in DNA isolated from bound complex --residues involved in binding will not be modified in DNA isolated from bound complex 1
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1) Modify 32 P labeled DNA fragment with DMS or other base-specific modification chemical. 2) Incubate DNA with protein. 3) Run on native gel and isolate bound and unbound complex. 4) Purify DNA from complex and cleave with piperidine. Cleave modified DNA not incubated with protein as control. 5) Run cleavage products on denaturing polyacrylamide gel. Run DNA fragment subjected to sequencing as position control. 6) Compare G residues in minus protein and plus protein lanes. Modification Interference Method 2
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Labeled probe GA GG CCGG Show that G3 and G4 are required for protein binding + DMS GA GG CCGG GA GG CCGG GA GG CCGG GA GG CCGG GA GG CCGG X X X X X 1 3 4 7 8
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This note was uploaded on 04/06/2010 for the course BCH BCH 453 taught by Professor Heminway during the Spring '10 term at N.C. State.

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lecture13 - Which nt Residues are Contacted by the Protein?...

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