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ChIP is a powerful tool for the study of protein/DNA interactions because it combines the specificity of immuno-precipitation, the sensitivity of PCR and the power of array profiling1,2,. In ChIP, intact cells are fixed using formaldehyde, which cross-links and preserves protein/DNA interactions at that precise time point. The DNA is then sheared into small, uniform fragments using either sonication or enzymatic digestion and specific protein/DNA complexes are immunoprecipitated using an antibody directed against the DNA-binding protein of interest. Following immuno-precipitation, cross-linking is reversed, the proteins are removed by treatment with Proteinase K and the DNA is recovered. The DNA is then analyzed, most commonly by PCR, to determine which DNA fragments were bound by the protein of interest (Figure 1).
While it is a powerful technique, performing ChIP is a complicated, multi-day procedure that is not well suited for the analysis of numerous samples. Moreover, troubleshooting the procedure and interpreting ChIP results can be difficult without validated reagents and controls that have been proven to work in ChIP.
CHIP-IT Express Improves Traditional ChipAll standard ChIP methods utilize agarose-based affinity beads to capture the antibody/chromatin complexes. However, spin-washing these beads is slow and tedious. In addition, DNA isolated through these methods must be further purified (by spin columns or phenol/chloroform extraction & precipitation) prior to PCR analysis.
We have developed a greatly simplified ChIP protocol that utilizes magnetic protein G-coated beads for antibody capture. Magnetic beads enable a streamlined ChIP protocol that requires little hands-on time and is compatible with multi-channel pipetting, so that many samples can be assayed simultaneously (Figure 2).
Reduced Background Eliminates Steps
The ChIP-IT™ Express method reduces or eliminates several time-consuming steps when compared with traditional ChIP (Table 1). The magnetic beads have much lower background than traditional agarose beads, so pre-clearing and blocking of the chromatin are no longer necessary. Wash steps are faster and fewer because centrifugation is replaced by magnetic pull-down, significantly reducing the amount of hands-on time. In addition, improvement of the chromatin elution and proteinase K treatment steps has completely eliminated the need for a separate DNA purification step after ChIP is complete. This not only saves time, it minimizes manipulations and eliminates DNA loss that occurs during purification, both of which help ensure sample-to-sample consistency.
Conclusions
ChIP-IT Express Kits transform ChIP from a labor-intensive protocol that is appropriate for only a small number of samples into a rapid method that is suitable for experiments requiring multiple samples (e.g. studies of pathway induction, time-course experiments, etc.). The kits incorporate all of the improvements described above and include the reagents required to perform your choice of either enzymatic- or sonication-type chromatin shearing, and perform 25 ChIP reactions, including a powerful bar magnet. For complete details, and to download the comprehensive protocol, please visit www.activemotif.com/chip.
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