Journal: Nucleic Acids Research
Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Figure Lengend Snippet: Schematic overview for the protein attached chromatin capture (PAtCh-Cap) method. Streptavidin coated magnetic beads are first conjugated with amine-PEG3-biotin (where PEG is polyethylene glycol). After standard cross-linking, chromatin isolation and DNA shearing procedures, 10% of the sample volume is removed and incubated with the pre-conjugated beads in the presence of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride). EDC reacts with protein carboxylate groups, forming unstable o -acylisourea activated esters that can undergo nucleophilic attack by the primary amines on the amine-PEG3-biotin prosthetics. This forms a covalent amide linkage with proteins in the chromatin complexes and releases an isourea by-product. Once bead-bound, these chromatin complexes can be subjected to additional chemical processing steps in parallel with bead-bound chromatin that was separately immunoprecipitated for the target of interest.
Article Snippet: Following two series of washes with 0.01 M PBS (pH 7.4), 50 μg of M-280 streptavidin coated Dynabeads (Thermo Fisher Scientific; equivalent to the number of beads utilized per reaction in the Active Motif ChIP-exo Kit) were conjugated with 10 μl of 50 nM EZ-link amine-PEG3-Biotin (Thermo Fisher Scientific; PEG = polyethylene glycol) in 0.01 M PBS (pH 7.4) at room temperature for 20 min.
Techniques: Magnetic Beads, Isolation, Incubation, Immunoprecipitation