By Abelson J.N., Simon M.I., Sauer R.T.
Tools familiar within the examine of DNA binding proteins are awarded during this quantity. those comprise purification and protein characterization, assays of protein-DNA binding and protein-induced bending, and biochemical and genetic tools for probing the constitution, strength, and specificity of protein-DNA interactions.
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Additional resources for Protein - DNA Interactions
Heat at 65 ° for 15 min to inactivate the kinase. Let the sample cool to room temperature, and then add 750 /zl ethanol. Mix by inversion. Spin (microcentrifuge) for 15 min at room temperature to pellet DNA. Discard supernatant. 4. Add 225/xl TE. Dissolve the pellet by vortexing. Then add 250/zl phenol-chloroform (1 : 1, v/v). Vortex for 1 min. Spin 5 min to separate phases. Transfer the upper layer to a new tube. Then add 250/zl chloroform-isoamyl alcohol (24 : 1, v/v). Vortex for 1 min. Spin 5 min to separate phases.
48, 507 (1987). e e j. -X. Hofmann, T. Laroche, A. H. Brand, and S. M. ) 57, 725 (1989). g A. R. Buchman, N. F. Lue, and R. D. Kornberg, Mol. Cell. Biol. 8, 5086 (1988). gg K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263, 17270 (1988). hh M. Cai and R. W. Davis, Mol. Cell. Biol. 9, 2544 (1989). 14 PURIFICATION AND CHARACTERIZATION  the preparation of partially purified protein fractions that are suitable for affinity chromatography. General Approach to Purification of Sequence-Specific DNA-Binding Proteins A strategy for the purification of sequence-specific DNA-binding proteins is as follows.
147, 11 (1981). 10 j. Greenblatt and J. ) 24, 421 (1981). 1i M. Sopta, R. W. Carthrew, and J. Greenblatt, J. Biol. Chem. 260, 10353 (1985). 26 PURIFICATION AND CHARACTERIZATION  In addition, the commercial availability allows good reproducibility and convenience. Preparation of Protein Ligand Ideally, the protein ligand that is used for affinity chromatography should be pure in order to ensure that every interacting protein that is detected is binding to the ligand rather than to a contaminant in the preparation.