TROUBLESHOOTING
Problem: How much tissue (and which kind) must be used to purify enough nuclei for a ChIP experiment?
[Step 1]
Solution: This protocol works well on tissues, such as liver, brain, lung, and placenta, and also on early mammalian embryos. In our laboratory, for instance, we have performed studies on 8.5-9.5 d.p.c. mouse embryos and placentas (Umlauf et al. 2004). Some 60 dissected embryos were used for each ChIP experiment. No more than ~0.2 g of tissue should be used for the volumes and tube sizes indicated in the protocol.
Problem: How can the yield of intact nuclei be maximized?
[Steps 4 and 12]
Solution: At Step 4 (or 12) of the nuclei purification procedure, it is critical not to extend the incubation in nuclei preparation buffer II for >10 minutes. For that reason, Step 5 should be initiated after 5 minutes of incubation in order to commence centrifugation (Step 6) at exactly 10 minutes after the addition of nuclei preparation buffer II in Step 4. Longer incubations can greatly reduce the yield of intact nuclei. For many tissues (liver, kidney, placenta), a final concentration of 0.2% of the nonionic detergent IGEPAL CA-630 (in nuclei preparation buffer II) will be enough to lyse the cellular membranes during the 10-minute incubation. However, we recommend testing 0.4% IGEPAL CA-630 for other tissues. For instance, this higher concentration of detergent slightly improves the yield of nuclei from brain and muscle tissues.
Problem: Is it a problem if the top layer (containing the IGEPAL CA-630) comes into contact with the nuclei pellet?
[Steps 7 and 15]
Solution: At Step 7 (or 15), it is essential that no traces of the top layer (containing the IGEPAL CA-630) come into contact with the nuclei pellet (even small traces of IGEPAL CA-630 may aberrantly affect the subsequent digestion of chromatin by MNase). Usually, we remove the top layer and the sucrose cushion from the tube by using Pasteur pipettes. This is done by aspirating from the surface of the solution, while changing the Pasteur pipette very often. If the top layer nevertheless comes in contact with the nuclei pellet, the pellet should be gently rinsed once with 1 ml of nuclei preparation buffer III before proceeding with Step 8 (or 16).
Problem: What can be done if, after the MNase digestion, the chromatin appears to be digested too much or too little?
[Step 43]
Solution: Fractionation of chromatin depends on the batch of MNase used, the concentration of the nuclei in the tube, the time of incubation, and the tissue type from which the nuclei were purified. If one observes too much digestion of the chromatin (i.e., almost all chromatin is digested to mono- and dinucleosome fragments), a lower concentration of MNase should be used. Inversely, in case little material is obtained in the S1 fraction, the amount of enzyme should be increased.
Problem: What can be done to improve ChIP when using chicken antisera that do not bind well to either protein A or protein G?
[Step 47]
Solution: We recommend adding 5 µg of a rabbit anti-chicken antiserum directly after Step 47 for a second precipitation of 3-4 hours, before proceeding with the extraction of the antibody-bound chromatin.
Problem: When an antiserum is used for the first time, how does one verify that the histone modification it is directed against has become enriched in the antibody-bound fraction?
[Step 69]
Solution: This can be done by purifying the histone proteins from the antibody-bound fraction (from Step 69), followed by electrophoresis through acid-urea-Triton gels. After electrophoresis, proteins are Western-blotted to nylon filters, which are immunostained with the antiserum following standard procedures (Gregory et al. 2001).
