Chromatin immunoprecipitation (ChIP) is an immunoprecipitation technique ChIP is based on the use of antibodies to study specific protein-DNA interactions. Antibodies are used to immunoprecipitate the protein of interest and its associated DNA. This powerful method provides valuable information about the interaction between certain proteins and specific DNA sequences, as well as insight into the regulation of gene expression.
Creative Biolabs provides an optimized high-performance solution to meet ChIP. We describe a useful ChIP protocol that includes complete reagents, detailed steps and a troubleshooting guide for optimizing experiments. You can browse the information below to learn more about how to optimize ChIP.
|Stages||Solutions and Reagents|
|Sample Preparation||Phosphate buffer (PBS), crosslinking agent, termination buffer, lysis buffer|
|Immunoprecipitation||Dilution buffer, antibody solution, magnetic or agarose beads|
|Washing and Elution||Washing buffer, elution buffer, protease|
ChIP involves a variety of proteomics and molecular biology methods. It involves cross-linking of proteins to DNA, fragmentation, preparation of soluble chromatin and immunoprecipitation with antibodies that recognize the target protein. The following steps describe the details.
The use of crosslinkers penetrates into the cell and locks the protein and DNA together. In vivo crosslinking has traditionally been achieved with formaldehyde. Of course, you can also choose other suitable crosslinkers depending on the sample. Dilute the cells and incubate with the crosslinking agent to crosslink the protein-DNA complex. Then add termination buffer to quench the crosslinker. Shake and mix to precipitate the cells, and finally remove the medium.
Add lysis buffer to resuspend the cell precipitate. Pipette up and down and incubate. Then sonicate the cell suspension and shear the chromatin to a suitable average length. Optimize sonication conditions according to different cell lines. After sonication, centrifuge the sample to precipitate cell debris. Finally, the supernatant is collected as the chromatin preparation to be subsequently analyzed.
Add dilution buffer to dilute the supernatant. Then add the antibody solution to the sample and incubate at room temperature. Then, you can add magnetic or agarose beads to the samples and immunoprecipitate them overnight on a rotating device.
If using magnetic beads, leave the tube in the magnet to collect the beads. If using agarose beads, collect the beads by centrifugation. Then perform several washes using washing buffer. After washing, then add elution buffer to the bead precipitate and slowly vortex to elute the complexes. Then centrifuge the sample and collect the supernatant.
The crosslinking between protein and DNA must be reversed prior to analysis of the specific DNA product. You can digest the proteins by thermal incubation or protease. Or use a DNA purification kit to clean up and concentrate the DNA preparation.
The DNA products obtained are usually measured by real-time quantitative PCR. Alternatively, sequencing can be used for analysis.
Below you will find some solutions that can be used to solve common problems observed in ChIP applications. We hope you will find this information useful.
No or low signal
ChIP is a powerful method for probing the interaction between proteins and DNA. Understanding and using the correct protocol and addressing common problems is critical to success. For comprehensive or personalized experimental resources, please contact us.
For research use only. Not intended for any clinical use.