✂️➕🏃🏽♀️ℹ️ Learn more about the benefits of the CUT&RUN assay: cst-science.co...
CUT&RUN is a low cell number alternative to chromatin immunoprecipitation. This animation describes how specific protein-DNA complexes are isolated to enable chromatin mapping.
Transcript:
Dynamic interactions between proteins and DNA drive many biological processes, including expression of genes that contribute to cell differentiation, development, and progression of diseases. Mapping where and when these protein-DNA binding events occur is essential for mechanistic studies of epigenetics in both normal development and disease.
To generate this kind of data, scientists routinely use chromatin immunoprecipitation. This involves a three-day-long protocol including steps for chromatin cross-linking and fragmentation, immunoprecipitation, isolation and elution of complexes, and DNA purification.
Now, a newer approach known as CUT&RUN is available. How does CUT&RUN work, and how is it different from ChIP? To prepare the input material for CUT&RUN, cells are harvested,
bound to magnetic beads to simplify handling and minimize sample loss,
and permeabilized with digitonin. No crosslinking fixative is needed, so the chromatin is kept in its native state.
Next, an antibody to the protein of interest is added to the permeabilized cells. The target may be a transcription factor, cofactor, or histone modification. The enzyme Protein A-Protein G Micrococcal Nuclease is added next, and is recruited to the chromatin by the interaction of the antibody with protein A or protein G. When calcium is added, the enzyme cuts the DNA at the site of the protein-DNA interaction. This design confers precise targeting and efficient control of nuclease activity. The complex containing the digested DNA is free to diffuse into the supernatant, while the remaining chromatin is trapped in the nuclei, simplifying subsequent DNA purification.
Compared to ChIP, CUT&RUN generates lower background, from less starting material, and requires fewer sequencing reads. CUT&RUN goes from cells to DNA in just one to two days. Inclusion of spike-in control DNA ensures experimental reproducibility.
CUT&RUN reagents and kits from Cell Signaling Technology® offer flexible options to fit your needs. For a complete solution, choose the CUT&RUN Assay Kit which comes with the required buffers, reagents, and controls, plus a detailed protocol. Just add a validated primary antibody against your protein of interest.
Or, you can choose the enzyme and spike-in DNA bundle, or individual reagents needed to complete your experiment.
To find kits, reagents, and antibodies for your CUT&RUN experiments, click here: cst-science.co...
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29 авг 2024
