Molecular interaction experiment: ChIP chromatin immunoprecipitation

1、 Technical Introduction

ChIP (Chromatin Immunoprecipitation) is a key technique for studying the interaction between proteins and DNA, widely used in fields such as transcription factor binding site identification and epigenetic modification analysis.

2、 Technical principles

By utilizing the specific binding of antigen antibodies, the DNA fragments bound to the target protein can be precipitated, which can truly reflect the regulatory proteins bound to the DNA sequence.

3、 Application scenarios

1. Transcriptional regulation research: Identification of transcription factors (such asThe DNA binding sites of p53 and NF - κ B.

2. Epigenetics: Analyzing histone modifications (such asThe role of H3K27me3 in gene silencing.

3. Disease mechanism: Explore the genomic binding patterns of abnormal proteins (such as MYC) in cancer.

4. Can be combined withThe combination of QPCR, Seq and other technologies.

4、 Technical advantages

ChIP captures the dynamic binding of proteins and DNA in the real state of cells through formaldehyde cross-linking, avoiding false negatives or false positives in vitro experiments, and is particularly suitable for studying transient binding events such as transcription factors.ButSpecific antibodies canagainAccurately enriching DNA fragments bound to target proteins, combined with qPCR or sequencing (ChIP seq) to achieve detection at the low to thousand cell level (such as optimizing reagent kits), with significant sensitivityAnd it has wide applicability.

5、 Sample type

Fresh cells (adherent cells/suspended cells), frozen cells, frozen tissues, and cross-linked cell samples.

6、 Sample requirements

Cell samples: approximately 1 × 10 ^ 7 (can be stored in a 10cm dish, suspended in a centrifuge tube, or in a T75 bottle);

Frozen tissue: approximately 70mg;

Cross linked cell samples: Cells were treated with 1% formaldehyde cross-linking.

7、 Sample preparation&transportation

Live cell samples: Fresh cell samples stored in dishes/T75 bottles/centrifuge tubes can be transported by express delivery at room temperature (with the latest delivery the next day) for those who are closer. Within the city, they can be delivered by flash or in person. For those who are farther away, cells can be frozen at -80 ℃ and transported by dry ice;

Organizational samples&cross-linked cell samples: frozen at -80 ℃ and transported in dry ice.

8、 Required reagents

Detect ChIP grade antibodies.

9、 Experimental cycle

1-2 weeks (excluding sequencing)

10、 Other

When selecting RT PCR for subsequent testing, the sites for subsequent testing must be provided.

Specific steps for formaldehyde crosslinking cells: ① Add 1% formaldehyde to the cell dish and incubate at 37 ℃ for 10 minutes; ② Wash the cells twice with cold DPBS containing [1mM PMSF, 1 μ g/ml aprotinin, and 1 μ g/ml pepstatin A]. ③ Scrape the cells into 2ml of cold DPBS containing protease inhibitors, transfer to centrifuge tubes, and freeze for shipment.