Cross-linking stabilizes the association of your target protein with its interacting DNA sequences. In some cases, target proteins are already tightly associated with DNA and additional chemical cross-links are not required to preserve the protein/DNA complex during the experimental analysis. This is called native ChIP, or N-ChIP. N-ChIP is suitable for targets such as histones and histone modifications. However, it is important to note that, without additional cross-linking, in vivo modifications in nucleosomes may still occur during your analysis. When targeting proteins that bind weakly to DNA, we highly recommend a crosslinking ChIP (X-ChIP) protocol. X-ChIP may be performed with UV light, formaldehyde, or other chemical cross-linkers.
Formaldehyde cross-linking is typically preferred for in vivo sample preparations, because this modification is reversible and will allow you to isolate and amplify your ChIP-enriched DNA. Additionally, the cross-linking distance of formaldehyde is only 2 Å (0.2 nm), ensuring that you are cross-linking proteins that are already closely associated with DNA. Formaldehyde will also form cross-links between DNA-binding proteins and proteins associated with them, facilitating the study of indirect protein/DNA interactions. Formaldehyde cross-linking is able to withstand subsequent experimental manipulations, allowing you to isolate intact protein/DNA complexes. However, formaldehyde may sometimes produce nonspecific cross-linking.
Cell lysis facilitates the release of the cell nuclei and eliminates components of the cytoplasm that can contribute to background signals. The success of your ChIP assay will depend on the effectiveness of the lysis procedure and the final recovery of chromatin. Some ChIP protocols specify the use of lysis buffers with high concentrations of sodium dodecyl sulfate (SDS), which allows whole cell lysis, whereas others specify the use of buffers for nuclei isolation. Lysis is achieved by incubation in appropriate concentrations of detergent-based buffers and protease inhibitor cocktail. It is also recommended that you apply mechanical force with a dounce homogenizer, mini- bead beater, vortexer, or glass beads. The method you choose will depend on the type of cells or tissues you are processing. If cell lysis is especially challenging for you, it may be helpful to view the sample before and after lysis under a phase contrast microscope. This will allow you to determine whether the nuclei have been released from your cells, or if using a whole cell lysis buffer, if the entire cell and nuclear membranes have been disrupted.
For more information about ChIP experiment guides, troubleshooting tips and supplementary protocols, please view our Chromatin Immunoprecipitation (ChIP) page.