Various ChIP methods are filled with all sorts of secret tricks that don’t seem to make it into the textbooks. This is especially true of the chromatin fixation step, where loads of variables make it difficult to pin down the exact conditions you need.
Instead of learning the hard way, through failed experiments or apprenticing for years at the side of veteran ChIP practitioners, we talked with ChIP expert Hamid Khoja, Senior Applications Scientist at Covaris, who shared with us his hard-won insights about proper chromatin fixation that just might keep your next ChIP from the scrap heap.
Common Cross-Linking Issues
During the development of their chromatin shearing instruments and ChIP kits, Khoja and the Covaris team evaluated every ChIP protocol they could find, and in the process found some common problems that can have huge effects on ChIP analysis.
- Fixation Time- “Most protocols are developed for probe sonicators, which are very harsh on chromatin. As a result we found that people were fixing for way too long to compensate for that” Says Khoja. Excessive fixation times make it much more difficult to shear chromatin to a consistent and reproducible length; a critical step in downstream analysis methods like Next Generation Sequencing (NGS).
- Overheating the Sample- “Probe and bath sonicators expose the chromatin to large amounts of energy and heat, which then require longer fixation times in their protocols in order to maintain a degree of epitope integrity during this harsh sonication process. The downside of that extra heat energy in a sample is thermal damage to the epitopes, possible reversing of the cross links, or even fragmentation of the fixed proteins thereby losing the epitope.” Khoja commented.
- Fresh Reagents- The components of your fixing agent can also have a big impact on your ChIP success. Case in point; the age and methanol content of formaldehyde. The effective concentration for formaldehyde drop significantly over time as bottles are exposed to air and light. This can have a significant effect on reproducibility of chromatin cross-linking and subsequent shearing, IP, and overall results. Additionally, some formaldehydes contain methanol as a preservative. Methanol, however, increases the permeability of cells, thereby increasing the efficiency of fixation. As Khoja explains, “A lot of formaldehyde that is used has up to 15% methanol. When people use this type of composition, they’re effectively fixing for a much longer time. Our titration experiments on methanol concentration showed that even at 1% methanol, the samples were over cross linked in 5 minutes as compared to formaldehyde without methanol.”
Get a Good Fixation
Here are some helpful tips from the Covaris crew to combat those previously mentioned issues and help you develop your own chromatin fixation (in a totally healthy way):
- Try an isothermal shearing method if possible. This does less damage to the chromatin and requires less fixing.
- Use only methanol-free formaldehyde sold in single use ampoules, and replace your stock every month. This will prevent over-fixation and increase the reproducibility and efficiency of your IP.
- Watch the cross-linking temperature. “Fixation is diffusion dependent, so it is effected by temperature. Fixing for 5 minutes at room temperature is not the same as fixing for 5 minutes at 37C.” Cautions Khoja.
- Avoid fixing your cells in media since most of the formaldehyde will fix the proteins in the media rather than fixing the cells. Also, suspension cells fixed in media will aggregate causing a very wide range of fixation and effect downstream analysis.
- Carry out a fixation time course for your cell line to empirically determine the optimal fixation time for your cell line and epitope of interest. Cell lines and epitopes differ wildly in their fixation efficiency, and sensitivity to fixation.
If you want to get the most from your ChIP analyses, try integrating the techniques Covaris has passed along into your own ChIP protocols; or take full advantage of their expertise, and check out their optimized protocols for truChIP™ Chromatin Shearing, Kits and AFA™ Instruments today!