The SNAP-CUTANA™ DYKDDDDK Tag Panel of spike-in controls for CUT&RUN offers an in-assay control to validate anti-FLAG® antibodies and confirm the success of CUT&RUN reactions involving FLAG epitope-tagged chromatin proteins. This essential positive control guides troubleshooting to differentiate problems with FLAG epitope-tagging (including transgene expression, chromatin binding of the tagged protein, solvent accessibility of the tag, etc.) from technical failures in the CUT&RUN workflow. The panel consists of two nucleosomes containing unmodified histone H3 or 3xDYKDDDDK-H3 fusion, each wrapped with two uniquely barcoded DNA templates (A and B, for an internal technical replicate). The nucleosomes are individually conjugated to paramagnetic beads and pooled into a single panel for convenient one-step spike-in to CUT&RUN reactions. The panel is added alongside ConA-immobilized cells just prior to the addition of anti-FLAG or IgG negative control antibodies (see Application Notes and Table 1). The release of genomic chromatin and the barcoded nucleosomes by pAG-MNase is dependent on the specificity of the antibody used.
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