Question

You want to perform chromatin immunoprecipitation (ChIP) experiments for RNA polymerase II (RNAP2), specifically to tell the difference between RNAP2 bound to the promoter and RNAP2 that has escaped the promoter but has not yet begun to fully elongate (RNAP2 serine 5 phosphorylated). Describe two issues you might have to deal with when designing your experiment to obtain the type of data you desire. also provide a way you might fix these problems

Answer 1

The RNA polymerase II enzyme is an important enzyme which co-ordinates and regulates the process of transcription in eukartyotes and hence is involved in genetic expression. The structure of the enzyme can be studied using techniques like Immunoprecipitation.

The necessity of the ChIP protocol is to know how the binding of the transcrition factors occurs in the active state and how does the phosphorylation take place. For this purpose, phospho-specific antibodies are taken namely, IgM antibodies. At the tail end of the enzyme there are a multiple repeats of amino acids which get targeted for any kind of modifications. The antibodies will show activity when the Serine in the fifth position of the amino acid repeat is phosphorylated.

The following issues can result while designing the protocol which can be resolved too as given below:

1) Always remember to start with optimum amount of the starting material since, a lot of it can result in reduced cross-linking which is a major disadvantage. Maintain the quantity between 5x106 to 1x107 cells.

2)Once the crossinking of the chromatin DNA is inititated, it needs to be stopped too since,excessive crosslinking will result in poor recovery of DNA and also lead to the DNA being inaccessible for the antigens. To stop the crosslinking, glycine solution of an appropriate molarity is used. A concentration of 125mM is preferably used.

3)While the sonication is carried out, care should be taken and the nature of the cells should be considered because you would not want any destruction of cells. Hence, for softer cells and fragments of a large size, the time required for sonication should be reduced.

4)Protein beads should be added to the chromatin DNA so that any protein which is not of interest will not bind to the antibody being introduced by us. They will first itself get cleared by the beads being used. Hence, no non-specific interaction will occur and only the desirable antigen-antobody interaction will occur.