A commonly used technique in epigenetics is Chromatin Immunoprecipitation, or ChIP for short. This technique can show you whether a certain protein (e.g. transcription factor or histone modification) binds to DNA, when in its native conformation, namely chromatin.
Insightful, but difficult
This information can be very insightful, but difficult to obtain. Most protocols and suggestions posted on the web are aimed at performing ChIP on cultured cells. In my experience, this is not at all the same as performing ChIP on chunks of tissue.
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What to do if you want to know the interaction of a protein with DNA in tissue, instead of in cells? Stay with me, because I will share my experiences in optimising a ChIP protocol for tissues with you.
Preparing good chromatin is a crucial part of a ChIP experiment. Read on to find out what is important in doing so.
Bear in mind that this is not a complete protocol for chromatin preparation, so search elsewhere to find out about other steps and buffer compositions.
1. Keep your snap-frozen tissues cold at all times- this keeps the proteins in and around the chromatin in good shape.
2. Determine the optimal tissue mass (try up to around 30 mg) for your specific experiment, as cell density and abundance of your target protein will determine the strength of the ultimate ChIP signal at later stages.
All subsequent steps in chromatin preparation will depend on it, so keep tissue mass constant between experiments.
3. Fragment the tissue. You can grind it with mortar and pestle while using liquid nitrogen. But since liquid nitrogen makes chunks of tissue jump in unpredictable directions, instead you can chop it up whilst keeping it chilled on a metal lid, placed on dry ice. Some protocols suggest ‘cubes of 1 mm2’. If you manage to cut it that regularly, well done, but anything that comes close will do just as well.
4. To be able to study genomic regions bound to your protein, you need to crosslink the two. This is generally done with 1% formaldehyde. Many protocols suggest 10 minutes at room temperature, but the optimal time (and temperature- some fix at 4°C) needs to be determined for each tissue (and antibody used for immunoprecipitation).
5. Lysis of the cells within the tissue is crucial for success. Protocols exist which do this in two steps: cellular lysis followed by nuclear lysis. My protocol only had a single lysis step, combined with vigorous vortexing and several rounds of sonication to help the SDS-buffer do its work. If you need to get a lot of lysate, read up on the methods for large colume cell lysis.
6. I have come to think that sonication (called sonifying by some) is an art in itself. It is at least a very important step in chromatin preparation, and, depending on the type of tissue, essential to obtain sufficient lysis. I used an ancient probe sonication device, found covered in dust in a forgotten lab corner. A great machine, but only after I had replaced the probe: an eroded microtip is a less efficient tip and will not give you reproducible results. An eroded tip can easily be recognised by eye. Microtip probes are more powerful than most modern sonication baths, thus crucial for tough tissues such as muscles. Sonication heats up the sample, so you’ll need to cool it. Keeping the tube in ice with some ethanol, and limiting the length of the sonication bursts, works well. Repeat however many rounds of sonication (e.g. 10-15 sec each time) until your tissue is visibly solubilised.
7. The next crucial step is again sonication, this time to fragment the chromatin. You want to shear it to obtain stretches of DNA of generally between 200 and 1200 base pairs, with associated proteins. Vary the number of sonication bursts: you can take out a small sample and continue sonicating, again putting a small sample aside, etc. Be aware though that decreasing the volume too much will change sonication conditions. Check shearing efficiencyby reversing crosslinks, then precipitating and cleaning the DNA and checking on gel if your fragments are in the right range. If lysis was not sufficient, you will notice at this stage, as DNA will remain of high molecular mass irrespective of how much you sonicate in the second round.
Be prepared to try all this several times before obtaining consistently good results. Only then you are ready to proceed onto the actual immunoprecipitation experiment and find out whether the protein of interest binds to your favourite genomic locus.
Who has any other suggestions to add?
Feel free to contact me if you’d be interested in my full protocol.