Molecular Interaction Experiment: CLIP UV Gel Immunoprecipitation

1、 Technical Introduction

CLIP(Cross-linking immunoprecipitation， 交联Co-immunoprecipitation）By combiningUV crosslinkingUsing immunoprecipitation method to analyze the binding sites of protein RNA interactions.

2、 Technical principles

CLIP technology utilizes ultraviolet radiation to covalently crosslink RNA and RNA binding proteins (RBPs) within cells. Subsequently, RNA fragments bound to specific RBPs are enriched through immunoprecipitation, followed by reverse transcription and high-throughput sequencing to obtain RNA binding sites and sequence information of RBPs in vivo.

3、 Application scenarios

1. Protein RNA interaction research: Determine the RNA binding sites and binding sequences of specific proteins in vivo, and reveal the mechanism of proteins in post transcriptional regulation.

2. Disease related research: By comparing the RNA binding of RBP under normal and disease states, RNA binding events and regulatory networks related to disease occurrence and development are discovered.

3. Drug development: Provide new targets and mechanisms of action for drug development, helping to develop drugs targeting specific RNA binding proteins or RNA molecules regulated by them.

4. Bioinformatics analysis: Through bioinformatics analysis of CLIP seq data, predict the binding sites and binding patterns of RNA binding proteins, construct RNA regulatory networks, etc.

4、 Technical advantages

1. In situ detection in vivo: It can capture the interaction between proteins and RNA in living cells, truly reflecting the physiological state in the body.

2. High specificity and strict purification: Cross linked complexes can be purified by specific antibodies or labels to effectively remove non-specific bound RNA and proteins, improving the credibility of the results.

3. Combined with high-throughput sequencing: By combining with high-throughput sequencing technology, the RNA binding sites and sequences of RBP can be identified across the entire genome, providing rich information.

5、 Sample type

Fresh cells (adherent cells/suspended cells), cell samples after UV crosslinking.

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);

Cross linked cell sample: 254 nm, 150 mJ/cm ² standard UV cross-linked treated cells.

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 (latest next day delivery) for those who are close to each other, and can be delivered by flash delivery or personal delivery within the city;

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.