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.).

Figure 1 presents an overview of the protocol.
1. Preparation of solutions and buffers
<ol>
	<li>Protein lysis buffer: Prepare the protein lysis buffer following a previously published report7 (Table 1).</li>
	<li>Protein lysis buffer + protease inhibitor (PI): Add protease inhibitor (see Table of Materials) to the above-prepared buffer (step 1.1). Store at -20 &#176;C.</li>
	<li>Sample b...

Protein Interaction Analysis in HEK-293 Cells Using Co-Immunoprecipitation

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+polypeptide+marker+sequence+useful+for+recombinant+protein+identification+and+purification.">A short polypeptide marker sequence useful for recombinant protein identification and purification.</a> Nature Biotechnology. 6 (10), 1204-1210 (1988).</li><li>Gerace, E., Moazed, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+pull-down+of+proteins+using+anti-FLAG+M2+agarose+beads.">Affinity pull-down of proteins using anti-FLAG M2 agarose beads.</a> Methods in Enzymology. 559, 99-110 (2015).</li><li>Einhauer, A., Jungbauer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FLAG+peptide,+a+versatile+fusion+tag+for+the+purification+of+recombinant+proteins.">The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins.</a> Journal of biochemical and biophysical methods. 49 (1-3), 455-465 (2001).</li><li>Egusa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+activation+of+PPAR&#945;+maintains+thermogenic+capacity+of+beige+adipocytes.">Selective activation of PPAR&#945; maintains thermogenic capacity of beige adipocytes.</a> iScience. 26 (7), 107143 (2023).</li><li>Nagano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+classical+brown+adipocytes+in+the+adult+human+perirenal+depot+is+highly+correlated+with+PRDM16-EHMT1+complex+expression.">Activation of classical brown adipocytes in the adult human perirenal depot is highly correlated with PRDM16-EHMT1 complex expression.</a> PLoS One. 10 (3), e0122584 (2015).</li><li>Seale, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+brown+fat+determination+by+PRDM16.">Transcriptional control of brown fat determination by PRDM16.</a> Cell Metabolism. 6 (1), 38-54 (2007).</li><li>Kajimura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+brown+and+white+fat+gene+programs+through+a+PRDM16/CtBP+transcriptional+complex.">Regulation of the brown and white fat gene programs through a PRDM16/CtBP transcriptional complex.</a> Genes and Development. 22 (10), 1397-1409 (2008).</li><li>Shinkai, Y., Tachibana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H3K9+methyltransferase+G9a+and+the+related+molecule+GLP.">H3K9 methyltransferase G9a and the related molecule GLP.</a> Genes and Development. 25 (8), 781-788 (2011).</li><li>Able, A. A., Richard, A. J., Stephens, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF&#945;+effects+on+adipocytes+are+influenced+by+the+presence+of+lysine+methyltransferases,+G9a+(EHMT2)+and+GLP+(EHMT1).">TNF&#945; effects on adipocytes are influenced by the presence of lysine methyltransferases, G9a (EHMT2) and GLP (EHMT1).</a> Biology. 12 (5), 674 (2023).</li><li>Ohno, H., Shinoda, K., Ohyama, K., Sharp, L. Z., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EHMT1+controls+brown+adipose+cell+fate+and+thermogenesis+through+the+PRDM16+complex.">EHMT1 controls brown adipose cell fate and thermogenesis through the PRDM16 complex.</a> Nature. 504 (7478), 163-167 (2013).</li><li>Bian, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+establishing+a+protein-protein+interaction+network+using+tandem+affinity+purification+followed+by+mass+spectrometry+in+mammalian+cells.">Protocol for establishing a protein-protein interaction network using tandem affinity purification followed by mass spectrometry in mammalian cells.</a> STAR Protocols. 3 (3), 101569 (2022).</li><li>Cristea, I. M., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+protein+complexes.">Affinity purification of protein complexes.</a> Cold Spring Harbor Protocols. 2011 (5), (2011).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+control+of+beige+fat+biogenesis+by+PRDM16+stabilization.">Post-translational control of beige fat biogenesis by PRDM16 stabilization.</a> Nature. 609 (7925), 151-158 (2022).</li><li>Holden, P., Horton, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crude+subcellular+fractionation+of+cultured+mammalian+cell+lines.">Crude subcellular fractionation of cultured mammalian cell lines.</a> BMC Research Notes. 2, 243 (2009).</li><li>Yang, L., Zhang, H., Bruce, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+detergent+concentration+conditions+for+immunoprecipitation+(IP)+coupled+with+LC-MS/MS+identification+of+interacting+proteins.">Optimizing the detergent concentration conditions for immunoprecipitation (IP) coupled with LC-MS/MS identification of interacting proteins.</a> Analyst. 134 (4), 755-762 (2009).</li><li>Herrmann, C., Avgousti, D. C., Weitzman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+salt+fractionation+of+nuclei+to+analyze+chromatin-associated+proteins+from+cultured+mammalian+cells.">Differential salt fractionation of nuclei to analyze chromatin-associated proteins from cultured mammalian cells.</a> BIO-PROTOCOL. 7 (6), e2175 (2017).</li></ol>

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...

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 into three types: in vitro, in vivo, and in silico. Co-immunoprecipitation (co-IP), affinity chromatography, tandem affinity purification, protein arrays, phage display, protein fragment complementation, X-ray crystallography, and nuclear magnetic resonance spectroscopy have been used for in vitro PPI detection3. Among these methods, co-IP is widely used because of its simplicity.
The fusion tag FLAG consists of eight amino acids (AspTyrLysAspAspAspAspLys: DYKDDDDK), including an enterokinase cleavage site, and was specifically designed for immunoaffinity chromatography4. DYKDDDDK-tagged proteins are recognized and captured using an anti-DYKDDDDK antibody. Therefore, they are efficiently pulled down using DYKDDDDK binding agarose beads5 to confirm their binding to specific proteins in a simple manner. Immunoprecipitation can be performed in a variety of cells, and a wide range of PPIs can be confirmed using antibodies against the protein of interest. Immunoprecipitation and peptide elution with anti-DYKDDDDK agarose beads have been previously reported5.
Here, we provide a simple immunoprecipitation method in which a plasmid encoding a DYKDDDDK-tagged protein is transiently introduced into HEK-293 cells to confirm the association of two proteins of interest. Certain DYKDDDDK antibodies can bind to both the N-terminus and C-terminus of the fusion proteins but not others6. Therefore, to avoid confusion, the antibody that recognizes the tag fused to both N- and C-terminus should be chosen. When inserting an epitope tag, it may be possible to avoid conformational changes in the protein by inserting 3 to 12 base pairs between the epitope tag and the target protein. However, the inserted sequence should be a base pair in multiples of 3 to avoid frameshift.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA (pH8.0)</td>
			<td>Nippon gene</td>
			<td>311-90075</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 50 &#181;L</td>
			<td>Biorad</td>
			<td>4561034</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Tris/Glycine/SDS</td>
			<td>Biorad</td>
			<td>1610772</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-FLAG M2 Affinity Gel</td>
			<td>Sigma</td>
			<td>A2220</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG, HRP-Linked Whole Ab Sheep</td>
			<td>GE Healthcare</td>
			<td>NA931-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper M</td>
			<td>Sumitomo Bakelite</td>
			<td>MS-93170</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I Coat Dish 100 mm</td>
			<td>IWAKI</td>
			<td>4020-010</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693132001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX supplement</td>
			<td>Invitrogen</td>
			<td>10565042</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS (-)</td>
			<td>FUJIFILM Wako</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>FUJIFILM Wako</td>
			<td>072-00626</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>FUJIFILM Wako</td>
			<td>077-00735</td>
			<td></td>
		</tr>
		<tr>
			<td>HA-Tag (C29F4) Rabbit mAb #3724</td>
			<td>Cell Signaling</td>
			<td>C29F4</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli Sample buffer</td>
			<td>Bio-Rad Laboratories</td>
			<td>161-0747</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Bio-Spin Chromatography Columns</td>
			<td>Biorad</td>
			<td>7326204</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell for Mini Precast Gels</td>
			<td>Biorad</td>
			<td>1658004JA</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal ANTI-FLAG M2 antibody produced in mouse</td>
			<td>Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>FUJIFILM Wako</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1-FLAG-PRDM16</td>
			<td>This paper</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1-HA-EHMT1</td>
			<td>This paper</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1-vector</td>
			<td>This paper</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride</td>
			<td>PSI</td>
			<td>24765</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution</td>
			<td>FUJIFILM Wako</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene(10) Octylphenyl Ether</td>
			<td>FUJIFILM Wako</td>
			<td>168-11805</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene(20) Sorbitan Monolaurate</td>
			<td>FUJIFILM Wako</td>
			<td>167-11515</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Sepharose 4 Fast Flow Lab Packs</td>
			<td>Cytiva</td>
			<td>17061801</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tubes</td>
			<td>eppendorf</td>
			<td>30108442</td>
			<td></td>
		</tr>
		<tr>
			<td>ROTATOR RT-5</td>
			<td>TAITEC</td>
			<td>RT-5</td>
			<td></td>
		</tr>
		<tr>
			<td>skim milk</td>
			<td>Morinaga</td>
			<td>0652842&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stripping Solution</td>
			<td>FUJIFILM Wako</td>
			<td>193-16375</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Mini PVDF Transfer Pack</td>
			<td>Biorad</td>
			<td>1704156B03</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo System</td>
			<td>Biorad</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>USDA Tested Fetal Bovine Serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Veriblot</td>
			<td>Abcam</td>
			<td>ab131366</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Actin (13E5) Rabbit mAb&#160;#4970</td>
			<td>Cell Signaling</td>
			<td>4970S</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunoprecipitation with an anti-epitope tag affinity gel to study protein-protein interactions

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.

Author Spotlight: Unraveling the Molecular Mechanisms of Brown and Beige Adipocyte Regulation

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

The scope of our research encompasses exploring the molecular mechanisms that regulate the development and function of brown and beige fat. Trunks, friction factors and co-factors such as the PRDM 16 complex, NFIA, and PPR alpha ELK-1 complex that we have more recently found control brown and beige fat cell fate and function. In addition, immune cell adipocyte crosstalk have been identified as key players.To advance research in your field, we employ several cutting edge technologies. These include quantitative PCR and chromatin immunoprecipitation for analyzing changes in gene expression as well as call immunoprecipitation and mass spectrometry to investigate alterations in protein-protein interactions, PPIs, associated with differentiation into beige adipocytes. Despite following the standard protocol for immunoprecipitation, detecting PPIs often proves challenging.Protein degradation is the primary cause of this issue, which we must carefully avoid. This protocol is simpler and less expensive than other techniques for examining PPIs, and does not include freeze thawing in the process, thus reducing protein degradation as much as possible.

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...

Watch this Scientific Journal Video about Author Spotlight: Unraveling the Molecular Mechanisms of Brown and Beige Adipocyte Regulation at JoVE.com

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 ...