Molecular interaction experiment: IP/CO-IP immunoprecipitation

1、 Technical Introduction

IP (immunoprecipitation) and Co IP (immunoprecipitation) are core technologies for studying protein function and interactions, widely used in the fields of molecular biology and cell biology.

2、 Technical principles

IP interacts with target proteins through specific antibodiesCombining and using antibodies to isolate target proteins (i.e. antigens), in order to detect the expression of a certain proteinCO-IP, on the other hand, verifies protein-protein interactions based on IP. By capturing a protein (bait protein) and detecting the interacting protein (target protein) that binds to it,thusSpecific enrichment of target proteins studiedFurther investigate the proteins in the complex that interact with known proteins.

3、 Application scenarios

• IP: Enrichment of low abundance proteins for mass spectrometry identification (IP-MS) and verification of antibody specificity.

• Co IP: Study transcription factor complexes, kinase substrate interactions, and validate known interactions or discover new interaction partners (such as signaling pathway complexes).

4、 Technical advantages

• IP: High specificity, dependent on antibody antigen specific binding.

• Flexibility: Supports can be selected from magnetic beads (easy to operate, compatible with automation) or agarose (high binding capacity).

• Co-IP: In vivo authenticity: It studies protein interactions in the natural state of the body and can reflect relatively real protein interactions.

5、 Sample type

Fresh cells (adherent cells/suspended cells), frozen cells, frozen tissues, and lysate samples.

6、 Sample requirements

• Cell samples: approximately 5 × 10 ^ 6 (can be 2 6cm dishes, or stored in suspension in centrifuge tubes, or 2 T25 bottles);

• Frozen tissue: about 40mg; lysate sample: protein concentration not less than 2mg/mg, total protein content not less than 3mg.

7、 Sample preparation&transportation

• Live cell samples: Fresh samples can be provided for those that are closer, and fresh cell samples cultured in dishes/T25 bottles/centrifuge tubes can be transported by express delivery at room temperature (latest next day delivery). Within the city, they can be delivered by flash or in person. For those that are farther away, cells can be frozen at -80 ℃ and transported by dry ice;

• Organizational samples&lysate samples: Frozen at -80 ℃ and transported in dry ice.

8、 Required reagents

Detecting IB antibodies and IP antibodies

9、 Experimental cycle

1-2 weeks (excluding mass spectrometry)

10、 Other

Subsequent Western blot/protein mass spectrometry analysis