EpiDyne-FRET Nucleosome Remodeling Assay Substrate Description: Mononucleosomes assembled from recombinant human histones expressed in E. coli (two each of histones H2A-Cy5, H2B, H3.2 and H4; accession numbers: H2A-P04908; H2B-O60814; H3.2-Q71DI3; H4-P62805) wrapped with a 207 base-pair 5' Cy3-conjugated DNA sequence that includes the Widom 601 nucleosome positioning element, with an added GATC motif. H2A has a Thr to Cys substitution at residue 120, the cysteine where Cy5 is conjugated. H3.2 has a Cys to Ala substitution at residue 110.
EpiDyne-FRET Nucleosome Remodeling Assay Substrate Formulation: Purified recombinant mononucleosomes, 50 µg (DNA + protein weight) in 10 mM Tris-HCl pH 7.5, 25 mM NaCl, 1 mM EDTA, 2 mM DTT, 20% glycerol. MW = 242,900 Da.
EpiDyne-FRET Nucleosome Remodeling Assay Substrate Storage and Stability: Stable for six months at -80°C from date of receipt. For best results, aliquot and avoid multiple freeze/thaws.
EpiDyne-FRET Nucleosome Remodeling Assay Substrate Application Notes: This product is a template for nucleosome remodeling enzymes (e.g.ACF Remodeling Enzyme Complex) using Cy3/Cy5 FRET, or using the restriction enzyme DpnII to determine accessibility of GATC which is masked in its native configuration (prior to remodeling). EpiDyne-FRET Nucleosome Remodeling Assay Substrate do not contain free DNA which could alter assayed activities.
EpiDyne-FRET Nucleosome Remodeling Assay Substrate References: Shahian T and GJ Narlikar (2012). Methods Mol Biol 833: 337-349.
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View FRET-based Chromatin Remodeling Assay application technical note for this product.
DNA Gel Data:Free DNA and Mononucleosomes run on a 6% polyacrylamide gel and stained with ethidium bromide to visualize DNA. Lane 1: Free DNA (Cy3-ST601-GATC1 DNA, 200 ng). Lane 2: Intact EpiDyne-FRET nucleosomes (370 ng). Note absence of free DNA in "Nuc", indicating all DNA complexed with histones.
Protein Gel Data: Coomassie stained PAGE gel of proteins in EpiDyne™ Nucleosome Remodeling Assay Substrate ST601-GATC1 (FRET) to demonstrate histone purity. Sizes of the histones are indicated. N.B. Cy5 conjugation decreases mobility of H2A.
Nucleosome Remodeling Data: RSC/ATP-dependent chromatin remodeling reaction. EpiDyne-FRET nucleosomes (20 nM) were incubated with RSC chromatin remodeler in the presence or absence of 2 mM ATP/ATPγS. Upon the addition of ATP, reactions were immediately read in an Envision Multilabel plate reader (PerkinElmer). Data are presented as the mean ±SD of the Cy3/Cy5 ratio (N=6).