FAQ of ChIP-zoonbio

①
When doing the ChIP experiment, everyone said that we should do a good job in the design of the control experiment, so how to design the control experiment?

The results of ChIP experiment are easy to be affected by many factors, such as the number of cells, the length of cross-linking time, the size of digestive fragments, the type of antibodies, etc., so when doing ChIP experiment, we must do a good job of experimental control, otherwise it is difficult to judge the reliability of the experimental results. After chromatin breaks, a portion of chromatin solution must be retained in a certain proportion as Input DNA for internal control： When the target antibody precipitates the protein DNA complex, a positive antibody and a negative antibody control should be set up at the same time. Only by comparing the results of the target antibody with the results of the positive and negative antibodies can we get a correct conclusion. The positive antibody usually selects the more conservative protein antibody which combines with the known sequence, usually the histone antibody or RNA Polymerase two antibody and so on. Negative antibodies usually select the serum of the target protein antibody host.

②
In the experiment, as well as the input comparison mentioned in the previous question, how does it come from and where is its importance?

Before immunoprecipitation, a part of the chromatin after fracture was taken as input control. Input is broken the split genomic DNA, together with the precipitated sample DNA, needs to undergo reverse cross-linking, DNA purification and final PCR or other methods for detection. Input control can not only verify the effect of chromatin fracture, but also convert the efficiency of chip according to the sample proportion according to the content of target sequence in Input and the content of target sequence in chromatin precipitation, so input control is an essential step in chip experiment.

③
DNA is best broken into 150-1000 BP fragments, but sometimes it is detected that the chromatin is too long or too short, such as more than 1000 BP or less than 100 bp. What is the reason and how to solve it?

If the cross-linking time is too long and the ratio of cells to Mn enzyme is too large, the chromatin fragment will be too long. At this time, the formaldehyde cross-linking time needs to be shortened. The cross-linking time is generally controlled at 5-60min. If the cross-linking time is too long, the chromatin of cells is hard to break, resulting in the fragment too long, which affects the results of ChIP. Moreover, the experimental materials are easy to be lost in the heart. At the same time, we also need to optimize the digestion steps of Micrococcal Nucleas, increase the amount of enzymes or reduce the number of cells. On the contrary, if the ratio of cells to Micrococcal Nucleas is too low, the fragment will be too short.

④
If we want to get a good result, what should we pay attention to in the selection of antibody?

The antibody of the target protein selected by chromatin immunoprecipitation is the key to the success of ChIP experiment. Because when protein and chromatin cross-linked, the epitope of antibody may be too close to the binding site to be recognized by antibody, so it can not effectively form immune precipitation complex in vivo, which directly affects the results of ChIP. So not all antibodies can do ChIP experiments. Only the antibodies verified by ChIP experiments can ensure the reliability of the experimental results.