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Protein-protein interactions are important for elucidating the function of target proteins, and co-immunoprecipitation (co-IP) can easily confirm PPIs. We transiently transfected a plasmid encoding an epitope-tagged protein into HEK-293 cells and developed an immunoprecipitation method to easily confirm the binding of two target proteins.
Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.
Proteins play a major role in all cellular functions, including information processing, metabolism, transport, decision-making, and structural organization. Proteins mediate their functions by interacting physically with other molecules. Protein-protein interactions (PPIs) are important for mediating cellular functions, such as mediating signal transduction, sensing the environment, converting energy into physical movement, regulating the activity of metabolic and signaling enzymes, and maintaining cellular organization1. Thus, PPIs can be used to elucidate unknown functions2. Methods for detecting PPIs can be classified....
Figure 1 presents an overview of the protocol.
1. Preparation of solutions and buffers
Thermogenic adipocytes, also known as brown and beige adipocytes, have potential anti-obesity and anti-glucose intolerance effects. PR (PRD1-BF1-RIZ1 homologous) domain-containing 16 (PRDM16) is a transcription cofactor that plays an important role in determining thermogenic adipocyte identity9,10.
EHMT1 (euchromatic histone-lysine N-methyltransferase 1), also known as GLP, primarily catalyzes the mono- and dimethylation of ly.......
This protocol is almost like previously reported protocols5,7,14,15. The important point of this protocol is that we never stop the experiment from the cell lysis step to the immunoprecipitation step. Protein degradation hinders PPI detection. Extended time course and repeated freeze-thaw cycles degrade proteins. Electrophoresis in SDS-PAGE should also be performed on the same day of immunoprec.......
This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 19K18008 (G.N.), JSPS KAKENHI Grant Number 22K16415 (G.N.), JSPS KAKENHI Grant Number 22K08672 (H.O.), Japan Diabetes Society Research Grant for Young Investigators (G.N.), and MSD Life Science Foundation Research Grant for Young Investigators (G.N.).
....Name | Company | Catalog Number | Comments |
0.5 M EDTA (pH8.0) | Nippon gene | 311-90075 | |
10% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 50 µL | Biorad | 4561034 | |
10x Tris/Glycine/SDS | Biorad | 1610772 | |
ANTI-FLAG M2 Affinity Gel | Sigma | A2220 | |
Anti-Mouse IgG, HRP-Linked Whole Ab Sheep | GE Healthcare | NA931-1ML | |
Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey | GE Healthcare | NA934-1ML | |
Cell Scraper M | Sumitomo Bakelite | MS-93170 | |
Collagen I Coat Dish 100 mm | IWAKI | 4020-010 | |
cOmplete, EDTA-free Protease Inhibitor Cocktail | Roche | 4693132001 | |
DMEM/F-12, GlutaMAX supplement | Invitrogen | 10565042 | |
D-PBS (-) | FUJIFILM Wako | 045-29795 | |
Glycerol | FUJIFILM Wako | 072-00626 | |
Glycine | FUJIFILM Wako | 077-00735 | |
HA-Tag (C29F4) Rabbit mAb #3724 | Cell Signaling | C29F4 | |
Laemmli Sample buffer | Bio-Rad Laboratories | 161-0747 | |
Micro Bio-Spin Chromatography Columns | Biorad | 7326204 | |
Mini-PROTEAN Tetra Cell for Mini Precast Gels | Biorad | 1658004JA | |
Monoclonal ANTI-FLAG M2 antibody produced in mouse | Sigma | F3165 | |
NaCl | FUJIFILM Wako | 191-01665 | |
pcDNA3.1-FLAG-PRDM16 | This paper | N/A | |
pcDNA3.1-HA-EHMT1 | This paper | N/A | |
pcDNA3.1-vector | This paper | N/A | |
PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride | PSI | 24765 | |
Penicillin-streptomycin solution | FUJIFILM Wako | 168-23191 | |
Pierce BCA Protein Assay Kit | Thermo scientific | 23227 | |
Polyoxyethylene(10) Octylphenyl Ether | FUJIFILM Wako | 168-11805 | |
Polyoxyethylene(20) Sorbitan Monolaurate | FUJIFILM Wako | 167-11515 | |
Protein G Sepharose 4 Fast Flow Lab Packs | Cytiva | 17061801 | |
Protein LoBind Tubes | eppendorf | 30108442 | |
ROTATOR RT-5 | TAITEC | RT-5 | |
skim milk | Morinaga | 0652842 | |
Stripping Solution | FUJIFILM Wako | 193-16375 | |
Trans-Blot Turbo Mini PVDF Transfer Pack | Biorad | 1704156B03 | |
Trans-Blot Turbo System | Biorad | N/A | |
Trizma base | Sigma | T1503-1KG | |
USDA Tested Fetal Bovine Serum (FBS) | HyClone | SH30910.03 | |
Veriblot | Abcam | ab131366 | |
β-Actin (13E5) Rabbit mAb #4970 | Cell Signaling | 4970S |
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