CUT&Tag Assay Kit #77552
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Product Information
Storage
All components in this kit are stable for 6 months when stored at the recommended temperature.
Protocol
Product Description
The CUT&Tag Assay Kit is designed to conveniently provide reagents needed to perform up to 24 reactions. The kit has been optimized to work in fresh or lightly fixed cells for all types of DNA-binding proteins, including histones, transcription factors, and cofactors. In addition, the kit has been optimized to work for histones in fresh or lightly fixed tissues. For analysis of transcription factors and cofactors in tissues, we recommend using the CUT&RUN Assay Kit #86652. If possible, we recommend using 100,000 cells or 1 mg of tissue per CUT&Tag reaction. If starting cell number is limited, this kit is validated to work with as few as 5,000 to 10,000 cells per reaction for histone modification targets and as few as 20,000 cells per reaction for transcription factors and cofactors. This kit is compatible with both whole cells and nuclei as starting material. We have not found that using nuclei generates a stronger signal or a higher signal-to-noise reaction compared to whole cells. A complete assay can be performed in as little as one day. The CUT&Tag Assay Kit also provides important controls to ensure a successful CUT&Tag experiment, including positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and negative controls Normal Rabbit IgG #2729 and Normal Mouse IgG #68860. The negative control Normal Rabbit IgG #2729 or Normal Mouse IgG #68860 is optional for the experiments, depending on the requirements of the peak calling algorithm used. This kit is compatible with downstream Next Generation sequencing (NG-seq) analysis, but not qPCR.
Specificity / Sensitivity
The CUT&Tag Assay Kit can be utilized with any CUT&Tag-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells (see Figures 1–3). One CUT&Tag reaction can use between 5,000 to 250,000 cells or 1 to 5 mg of tissue of starting material (see Figures 5-7). The kit is compatible with multiple species of antibodies, including rabbit and mouse (see Figure 4). The positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 detects multiple species of tri-methyl histone H3 Lys4 protein, including human, mouse, rat, and monkey.
Background
Similar to Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag) is a powerful technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1-3). CUT&Tag has many of the same advantages as the CUT&RUN assay in that it provides a rapid, robust, and true low cell number protocol for detection of protein-DNA interactions in the cell. In addition, the CUT&Tag assay adds an in situ adaptor DNA ligation step carried out by the pAG-Tn5 enzyme, in which an adaptor DNA is ligated directly to antibody-targeted chromatin DNA fragments in the cell. As a result, subsequent DNA library preparation is much faster and easier than library preparation following the CUT&RUN assay, free from DNA end repair, A-tailing, and adaptor ligation in vitro. CUT&Tag works very well for analyzing histone modifications, in addition to mapping some transcription factor and cofactor binding.
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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
CUT&Tag provided under a license from Active Motif, Inc. under U.S. Patent No. 10,689,643 and 9,938,524, foreign equivalents, and child patents deriving therefrom. For purchaser's internal research use only. May not be used for resale, services, or other commercial use.
U.S. Patent No. 11,733,248, foreign equivalents, and child patents deriving therefrom.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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