CUTANA™ Uncharged 6xHis-pAG-Tn5 for CUT&Tag
20 in stock
Powered by BiozProducts in EpiCypher’s IDEA Toolbox (Innovation and Discovery of Epigenetic Applications) offer access to reagents without known or fully defined uses, enabling researchers to explore cutting-edge applications. Due to their novelty and unexplored potential, EpiCypher will engage in limited technical support.
CUTANA™ Uncharged 6xHis-pAG-Tn5 is a fusion of proteins A and G to E. coli transposase (Tn5), the key enzyme for CUT&Tag [1]. The fusion protein contains a polyhistidine tag (6xHis) to enable applications that require affinity purification steps (for an untagged version of uncharged pAG-Tn5, see EpiCypher 15-1025). Uncharged Tn5 must be loaded with user-designed mosaic adapter DNA prior to use in CUT&Tag (see reference [1] and Application Notes).
His-pAG-Tn5 enables purification of differentially barcoded antibody-Tn5 complexes such as those described in MulTI-Tag (Multiple Target Identification by Tagmentation) and multi-CUT&Tag workflows to simultaneously interrogate multiple chromatin proteins in a single CUT&Tag reaction [2,3]. These approaches have been applied to map histone post-translational modifications and RNA Polymerase II down to single cell resolution to define cell-specific epigenetic landscapes.
Intellectual Property
Figure 1: Protein gel data
Figure 2: Functional validation in CUT&Tag
Figure 3: Functional validation of 6xHis binding
Various concentrations of CUTANA Uncharged 6xHis-pAG-Tn5 were incubated with biotinylated recombinant nucleosomes (rNuc; EpiCypher 16-0006). Uncharged pAG-Tn5 lacking a 6xHis tag (EpiCypher 15-1025) was used as a negative control. AlphaScreen technology (PerkinElmer/Revvity) was used to confirm functional 6xHis binding by using Nickel Chelate Donor Beads (Revvity AS101) to bind the 6xHis tag and Streptavidin Acceptor Beads (Revvity AL125) to bind biotin-rNuc. Signal (AlphaScreen counts) indicates 6xHis-pAG-Tn5 complexed with biotin-rNuc. No signal was observed in pAG-Tn5 lacking a 6xHis tag.
Figure 4: CUT&RUN genome-wide heatmaps
CUT&RUN was performed as described in Figure 5. Heatmaps show two replicates (“Rep”) of IgG (left) and H3K4me3 (right) kit control antibodies in aligned rows ranked by intensity (top to bottom) and colored such that red indicates high localized enrichment and blue denotes background signal.
- Type: Transposase
- Mol Wgt: 161,401.8 Da (dimer)
- Host: E. coli
- Epitope Tag: 6xHis
Storage
Stable for one year at -20°C from date of receipt. The protein is not subject to freeze/thaw under these conditions.
Formulation
50 mM HEPES-KOH pH 7.2, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% glycerol.
Application Notes
pAG-Tn5 transposomes can be assembled as previously described [1]. In brief, 3.2 µL of an equimolar mixture of pre-annealed user-defined Adapter-A and user-defined Adapter-B oligonucleotides (50 µM each, 100 µM total adapter DNA) should be mixed with 70 µL of 1.5 µM pAG-Tn5 fusion protein dimer (a 3:1 molar ratio of adapter DNA to pAG-Tn5 dimer). The mixture is then incubated on a rotating platform for 1 hour at room temperature and stored at -20°C. Specific activity definition of the charged pAG-Tn5 is highly recommended before use in CUT&Tag. Due to the confounding variable of user-supplied mosaic adapters, EpiCypher will not engage in protocol troubleshooting for this reagent.
| Item | Cat. No. |
|---|---|
| CUTANA™ 8-strip Tubes | 10-0009 |
| Magnetic Separation Rack, 0.2 mL | 10-0008 |
| Magnetic Separation Rack, 1.5 mL | 10-0012 |
| CUTANA™ Nuclei Extraction Buffer | 21-1026 |
| CUTANA™ Concanavalin A Conjugated Paramagnetic Beads | 21-1401/21-1411 |
| SNAP-CUTANA™ K-MetStat Panel | 19-1002 |
| Rabbit IgG Negative Control Antibody | 13-0042 |
| H3K4me3 Positive Control Antibody | 13-0060 |
| H3K27me3 Positive Control Antibody | 13-0055 |
| Anti-Rabbit Secondary Antibody for CUTANA™ CUT&Tag | 13-0047/13-1047 |
| Anti-Mouse Secondary Antibody for CUTANA™ CUT&Tag | 13-0048/13-1048 |
| CUTANA™ Non-Hot Start 2X PCR Master Mix for CUT&Tag | 15-1018 |
| CUTANA™ Quick Cleanup DNA Purification Kit | 14-0052 |
