Chromatin immunoprecipitation (ChIP) is the method used to study the interaction between DNA and protein in vivo. The principle lies in fixing the protein-DNA compound under the condition of living cells, randomly cutting into small chromatin fragments of a certain length and precipitating the compound with immunological methods for specific accumulating DNA fragments which can bind target proteins. The information on the interactions between protein and DNA is obtained by purification and detection of the target fragments.
ChIP can be used to detect the dynamic role of trans-acting factors and DNA in vivo and can be used in studying the relationship between various covalent modifications of histone proteins and gene expression. Moreover, ChIP by combining other methods has expanded its range of applications: ChIP-on-chip methods based on ChIP and gene chips have been widely used for high-throughput screening of specific trans-factor target genes; ChIP combined with in vivo footprinting Used to find in vivo binding sites for trans-factors; RNA-ChIP is used to study the role of RNA in gene expression regulation.
1. Formaldehyde treatment of cells.
2. Collection of cells, ultrasonic fragmentation.
3. Adding the target protein antibody for binding with the target protein-DNA compound.
4. Adding Protein A for binding with the antibody-target protein-DNA compound and precipitation.
5. Remove non-specific binding by washing the precipitation.
6. Elute to obtain the accumulated target protein-DNA compound.
7. Remove crosslinking for purification of the accumulated DNA fragments.
8. PCR or gene chip analysis.
2. Antibodies that qualified for ChIP: If to detect upon ChIP, take input as a control; if to perform q-PCR upon ChIP, provide the specific sequence at the predictive target gene binding site or the name, genus, sequence, or GeneBank ID of the target gene.
Experiment report: Detailed steps, sequencing results, q-PCR results
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