- How long does the lysate need to be sonicated for to shear the DNA to the appropriate size?
- How much protein is needed per IP?
- How much antibody is needed per IP?
- What is the importance of cross-linking?
- What is the optimal reverse cross-linking temperature and incubation time?
Optimal conditions to obtain the desired fragment size should be determined prior to the immunoprecipitation (IP) by performing a sonication time course. A recommended time course is for 5, 10, 15 and 20 min and the fragment size will decrease with an increased time course. Ensure that samples are kept ice cold throughout the sonication. Load 10 µl of sample on a 1.5 % agarose gel to analyze DNA fragment size. NOTE: sonicating for too long will disrupt nucleosome-DNA interactions therefore the band size should not be smaller than 200 bp.
Use approximately 25 μg of protein per IP. Protein concentration can be calculated using the Bradford assay. Dilute each sample 1:10 with RIPA Buffer.
The amount of antibody to be added has to be determined empirically. In the first instance try 3-5 μg of antibody per 25 μg of protein, this could be increased to 10 ug if no signal is observed.
A polyclonal antibody is preferable to a monoclonal; as monoclonal antibodies recognize only a single epitope, whereas within a polyclonal antibody population there will be a number of antibodies that recognize different epitopes. A polyclonal population will reduce the probability that all specific epitopes will be masked by the process of cross-linking, so there is a better chance of a positive result in X-ChIP.
Cross-linking is a time-critical procedure and should be carried out for 10 min. Excessive cross-linking can lead to a decrease in the amount of protein bound to the DNA and reduction in the availability of epitopes/changes in epitopes for antibody binding. In turn, this leads to reductions in the material bound/antigen available in the sample.
Cross linking is reversed using 5 M NaCl for 4 hours to overnight at 65 °C. It is important to perform this step for at least 4 hours.