Molecular experiment Molecular interaction: Pull down experiment

1、 Technical Introduction

Pull down experimentAlso known as pulldown experiment, it is an in vitro affinity purification technique.

2、 Technical principles

Fixing a protein onto a certain substrate (such asSepharose）However, when the cell extract passes through the matrix, ligand proteins that can interact with the fixed protein are adsorbed, while "impurities" that are not adsorbed flow out with the eluent. The adsorbed protein can be recovered by changing the eluent or elution conditions.

3、 Application scenarios

1. Verify known protein interactions: By conducting Pull down experiments on known bait proteins and prey proteins that may interact, if the presence of prey proteins can be detected, the interaction relationship between the two can be verified.

2. Screening for unknown protein interactions: Conduct Pull down experiments on bait proteins and cell lysate or other protein mixtures, and then identify the bound proteins to screen for unknown proteins that interact with bait proteins.

3. Study the composition of protein complexes: Separate protein complexes through Pull down experiments, and then identify the proteins in the complexes to determine their composition.

4. Study the interaction between proteins and nucleic acids: By using DNA Pull down and RNA Pull down techniques, the interaction between proteins and DNA/RNA can be studied, such as the binding of transcription factors to promoters, the binding of RNA binding proteins to RNA, etc.

4、 Technical advantages

It can be determined whether there is a direct interaction between the target protein and the detection protein.

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: approximately 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

If WB testing is to be performed, corresponding antibodies need to be provided.

9、 Experimental cycle

3-4 weeks (excluding mass spectrometry)

10、 Other

RNA-Pull-down、DNA-Pull-down、GST-Pull-down， Specific species and target proteins need to be informed, followed by Western blot/protein mass spectrometry testing. The cost of in vitro expression of the fusion protein needs to be evaluated and calculated separately based on the specific target protein.

Some experiments can also use recombinant expressed proteins for transcription factors.

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 400mg;

Plant tissue: about 2g; nuclear protein, purified transcription factors, recombinant expressed proteins, etc.: each membrane provides at least 50ul, with a concentration of not less than 0.5mg/ml.

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&other liquid samples: -80 ℃ frozen dry ice transportation.

8、 Required reagents

More than 50ul of protein specific antibodies are bound, with a concentration greater than 0.5mg/ml.

9、 Experimental cycle

2-3 weeks

10、 Other

If there is no probe sequence, please provide information or relevant literature on DNA binding proteins.