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The pAG-MNase enzyme is conjugated to an antibody for proteins of interest. Chromatin fragments containing proteins of interest can then be purified using immunoprecipitation techniques. After this, the DNA fragments are purified and sequenced. The sequencing results can be used to determine the regions of DNA your protein of interest interacts with.
As outlined in the protocol below, Abcam provides ChIC/CUT&RUN-seq validated antibodies and the complementing pAG-MNase enzyme.
Figure 1. Schematic of the ChIC/CUT&RUN protocol. Nuclei are attached to magnetic Concanavalin A beads to allow ease of handling and safe liquid removal after each wash step. Nuclei are permeabilized and simultaneously incubated with an antibody against the protein of interest. Protein A/G-MNase (ab285373) fusion protein binds to the antibody against the protein of interest. When Ca2+ is added, the MNase cleaves the DNA on both sides of the formed complex and releases DNA fragments that diffuse out of the nucleus. The DNA can be extracted and used in an end-repair and adapter ligation-based DNA library preparation. NGS informs the binding profile of the protein of interest via the frequency of sequences in a particular region.
Time: 1 h
1. Harvest cells and suspend in 3 – 4 mL ice-cold PBS.
2. Count cells and take the desired amount of cells per reaction.
3. Prepare wash buffer.
4. Wash cells three times in wash buffer.
5. Prepare a slurry of conA magnetic beads in binding buffer
6. Bind cells to activated beads
7. Separate the cell-bound beads from solution
Materials required:
Time: Overnight
1. Prepare the antibody buffer
2. Resuspend beads in antibody buffer
3. Add antibody to bead mix
You should use horizontal incubation to prevent the ConA beads from drying out.
Time: 1 h 15 min
1. Separate the antibody-bound beads from the solution.
2. Prepare Digitonin wash buffer.
3. Wash beads twice with digitonin wash buffer.
4. Incubate the beads with pAG-MNase solution.
You should use horizontal incubation to prevent the ConA beads from drying out.
Time: 40 min
1. Separate the antibody-bound beads from the solution.
2. Prepare low salt, incubation and wash buffers.
3. Wash beads twice with digitonin wash buffer.
4. Wash beads with low salt buffer.
5. Digest chromatin by incubating beads with ice-cold incubation buffer.
Time: 35 min
1. Incubate beads with stop buffer.
This is to stop the reaction and release the digested DNA fragments into solution.
2. Collect fragments.
The supernatant contains DNA fragments.
3. Extract DNA and prepare for sequencing.
4. Send DNA fragments for sequencing.
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