A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions.
Using the single cell eukaryote Tetrahymena thermophila, a simple method was developed for studying protein-DNA associations by cross-linking proteins to DNA with formaldehyde and immunoprecipitating the solubilized chromatin fragments with a specific antiserum. The protocol uses crude antiserum and involves only three steps: cross-linking, shearing to solubilize the chromatin, and immunoprecipitation. Methods for optimizing certain critical parameters, such as fixation time and NaCl concentration, are described. The method is likely to be generally useful for a variety of nuclear antigens.
Dedon
P C
PC
Department of Biology, University of Rochester, New York 14627.
Soults
J A
JA
Allis
C D
CD
Gorovsky
M A
MA
D000818:Animals; D002843:Chromatin / Q000378:metabolism; D003432:Cross-Linking Reagents; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D005404:Fixatives; D005557:Formaldehyde; D006609:High Mobility Group Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D011233:Precipitin Tests / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000378:metabolism
D002843:Chromatin; D003432:Cross-Linking Reagents; D005404:Fixatives; D006609:High Mobility Group Proteins; D006657:Histones; D011506:Proteins; D005557:Formaldehyde; D004247:DNA