A non-isotopic in vitro assay for histone acetylation.
We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH(2)-terminal tails of histones H3 and H4 linked to epitope-tagged maltose-binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions.
Kuninger
David
D
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Road, Mail Code L224, Portland, OR 97239, USA.
Lundblad
James
J
Semirale
Anthony
A
Rotwein
Peter
P
D000107:Acetylation; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D018604:Epitope Mapping; D006657:Histones / Q000737:chemistry*; D015151:Immunoblotting / Q000379:methods*; D007553:Isotope Labeling; D051379:Mice; D008809:Mice, Inbred C3H; D050880:p300-CBP Transcription Factors / Q000032:analysis*; D000099224:p300-CBP-Associated Factor
D006657:Histones; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
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