Abcam has answered some of the common questions asked by researches about best practice in experiments involving chromatin precipitation.
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How much time should the lysate be sonicated for to achieve DNA shearing to the required size?
Before immunoprecipitation, a sonication time course should be used to determine the conditions required to produce the required fragment size. The sonication time course should have intervals of 5, 10, 15, and 20 minutes, with fragment sizing decreasing over time. To analyze the DNA fragment size, 10 µL of sample should be loaded on 1.5% agarose gel.
Samples should be kept below freezing point throughout sonication. Excessive sonication results in disruptions in the interactions between DNA and nucleosomes. For this reason, the band size should not be less than 200 bp.
How much protein is required for each immunoprecipitation?
For each immunoprecipitation, around 25 µg of protein should be used. All samples should be diluted using a RIPA buffer to a factor of 1:10. Researchers can use a Bradford assay to determine the concentration of protein.
How much antibody is required for each immunoprecipitation?
The amount of antibody that should be added is determined through trial and error. For the first attempt, for every 25 µg of protein, 3 to 5 µg of antibody should be added. If no signal is observed this can be increased up to 10 µg.
Polyclonal antibodies are better to use than monoclonal antibodies. Within a single polyclonal antibody population there will be several antibodies which can recognize a variety of epitopes, whereas monoclonal populations will only recognize one. This means polyclonal populations provide a better chance of a positive X-ChIP result, as there is a lower chance of cross-linking masking epitopes.
Why is cross-linking significant?
The execution of cross-linking should be carried out for 10 minutes as it is time-critical. If cross-linking is too extensive the amount of protein-DNA binding is reduced which leads to a lower availability of epitopes for antibody binding.
The result of this is less available material bound/antigen in the sample.
What is the best incubation time and temperature for cross-linking?
Cross-linking reversals should take place for at least four hours at 65 °C in 5 M sodium chloride solution.
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