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 buffer: Mix 900 &#181;L of 4x Laemmli sample buffer (see Table of Materials) and 100 &#181;L of 2-mercaptoethanol. Store at -20 &#176;C.</li>
	<li>Phosphate-buffered saline (PBS) with polyoxyethylene (20) sorbitan monolaurate (PBS-P): Mix 1.998 L of PBS and 2 mL of polyoxyethylene (20) sorbitan monolaurate (see Table of Materials). Store at room temperature.</li>
	<li>1x Tris/Glycine/SDS buffer: Mix 450 mL of DDW and 50 mL of 10x Tris/Glycine/SDS. Prepare at room temperature on the day of use.</li>
</ol>
2. Plasmid transfection
<ol>
	<li>Culture HEK-293 cells in a 10 cm collagen-coated dish to subconfluence (80%-95%) using Dulbecco&#39;s modified eagle&#39;s medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (10,000 units/mL penicillin G and 10,000 &#181;g/mL streptomycin) (see Table of Materials).</li>
	<li>Prepare 1-10 &#181;g of plasmid7 to be transfected per dish and make a transfection mix. Add 150 mM NaCl to a final volume of 250 &#181;L per dish. Vortex and spin down the mixture.</li>
	<li>Add 60 &#181;L of 1 mg/mL Polyethylenimine (PEI, see Table of Materials)per dish, followed by 150 mM NaCl to a final volume of 250 &#181;L of PEI solution.</li>
	<li>Add the PEI solution to the transfection mix to make a mixed solution. Immediately vortex (at top speed for 3-5 s, perform the same thereafter) and spin down.</li>
	<li>Incubate at room temperature for 15-30 min. During this time, change the medium of HEK-293 cells.</li>
	<li>Sprinkle 500 &#181;L/dish of mixed solution all over the dish of HEK-293 cells and incubate overnight (13-14 h).</li>
	<li>Perform medium exchange the next day.</li>
</ol>
3. Cell lysis and sample preparation
NOTE: To avoid protein degradation, subsequent steps should be performed without preservation or freeze-thaw of the sample as much as possible.
<ol>
	<li>Approximately 48 h after transfection, wash the cells thrice with ice-cold PBS, add 500 &#181;L/dish of protein lysis buffer + PI, and collect the cells by cell scraper and a pipette in a tube with low protein adsorption on ice.</li>
	<li>Vortex every 5 min and incubate the samples on ice for 10 min.</li>
	<li>Centrifuge at 16,400 x g for 10 min at 4 &#176;C. Collect the supernatant and transfer it into a fresh 1.5 mL tube with low protein adsorption. The samples can be stored at -80 &#176;C.</li>
	<li>Determine the protein concentration of the samples with a protein concentration measurement kit according to the manufacturer&#39;s protocol (see Table of Materials).</li>
	<li>Separate 200-1000 &#181;g of the same amount of protein in each sample, then add protein lysis buffer + PI to adjust the total volume to 500-1000 &#181;L. 
	NOTE: The following steps are performed on ice.</li>
</ol>
4. Preparation of slurry
NOTE: Prepare a 1:1 slurry of protein G gel and epitope tag affinity gel the day before or on the day of immunoprecipitation.
<ol>
	<li>Preparation of a protein G gel
	<ol>
		<li>Using a tip with the end cut off, mix well and then separate the protein G gel (see Table of Materials) such that 20 &#181;L of beads per sample is prepared.</li>
		<li>Centrifuge at 12,000 x g for 20 s at 4 &#176;C and place the beads on ice for 1 min to allow the beads to level off. Discard the supernatant using a pipette.</li>
		<li>Add 1 mL of protein lysis buffer + PI to the protein G gel prepared in step 4.1.2 and mix by tapping and inverting. Centrifuge at 12,000 x g for 20 s at 4 &#176;C, place the beads on ice for 1 min to allow the beads to level off, and discard the supernatant.</li>
		<li>Repeat step 4.1.3 thrice.</li>
		<li>Add an equal volume of protein lysis buffer to the protein G gel to make a 1:1 protein G gel slurry. The protein G gel slurry can be stored at 4 &#176;C for approximately 1 day.</li>
	</ol>
	</li>
	<li>Preparation of an epitope tag affinity gel
	<ol>
		<li>Using a tip with the end cut off, agitate well and then separate the epitope tag affinity gel (see Table of Materials) such that 10-15 &#181;L of beads per sample is prepared.</li>
		<li>Centrifuge at 5,000 x g for 30 s at 4 &#176;C and wait for 1 min on ice to allow the beads to level off. Discard the supernatant using a pipette.</li>
		<li>Add 1 mL of protein lysis buffer + PI to the epitope tag affinity gel prepared in step 4.2.2 and mix by tapping and inverting. Centrifuge at 5,000 x g for 30 s at 4 &#176;C, place the beads on ice for 1 min to allow the beads to level off, and discard the supernatant with a pipette.</li>
		<li>Repeat step 4.2.3 twice.</li>
		<li>Add 500 &#181;L of 0.1 M glycine (pH 3.5) to the epitope tag affinity gel prepared in step 4.2.4 and mix by tapping and inverting. This step is performed to remove free epitope tag antibodies. Within 20 min after the addition of glycine, centrifuge at 5,000 x g for 30 s at 4 &#176;C, place the beads on ice for 1 min to allow the beads to level off, and discard the supernatant.</li>
		<li>Add 500 &#181;L of protein lysis buffer + PI to the epitope tag affinity gel prepared in step 4.2.5 and mix by tapping and inverting. Centrifuge at 5,000 x g for 30 s at 4 &#176;C, place the beads on ice for 1 min to allow the beads to level off, and discard the supernatant.</li>
		<li>Repeat step 4.2.6 thrice.</li>
		<li>Add an equal volume of protein lysis buffer to the epitope tag affinity gel to prepare a 1:1 epitope tag affinity gel slurry. The epitope tag affinity gel slurry can be stored at 4 &#176;C for approximately 1 day.</li>
	</ol>
	</li>
</ol>
5. Preclearing with the protein G gel and capturing protein complexes with the epitope tag affinity gel
<ol>
	<li>Mix 1:1 protein G slurry (prepared in step 4) with a pipette fitted with a cut tip and add 40 &#181;L at a time to the sample.</li>
	<li>Rotate the sample for 1 h on a rotator (see Table of Materials) in approximately 30 s per cycle in a refrigerated chamber (4 &#176;C).</li>
	<li>Centrifuge at 12,000 x g for 20 s at 4 &#176;C and place the beads on ice for 1 min to allow the beads to level off.</li>
	<li>Collect the supernatant and transfer it into a new 1.5 mL tube with low protein adsorption.</li>
	<li>Separate the sample for input from the sample in step 5.4 and transfer it into a new 1.5 mL tube.</li>
	<li>Add 20-30 &#181;L of 1:1 epitope tag gel slurry to the sample.</li>
	<li>Rotate on a rotator for approximately 30 s per cycle in a refrigerated chamber (4 &#176;C) for 2 h.</li>
	<li>Add sample buffer to the input prepared in step 5.5 to dilute it to 4x, and heat at 98 &#176;C for 10 min. The input can be stored at -80 &#176;C. 
	NOTE: Because of the possibility of protein degradation due to freezing and thawing, subsequent steps should be performed without preserving the protein as much as possible.</li>
</ol>
6. Washing and eluting the precipitated proteins
<ol>
	<li>Set the temperature of the heat block to 98 &#176;C.</li>
	<li>Centrifuge at 5,000 x g for 30 s at 4 &#176;C and wait for 1 min on ice to allow the beads to level off. Discard the supernatant using a pipette.</li>
	<li>Add 1 mL of protein lysis buffer + PI and mix by tapping and inverting. Rotate on a rotator for approximately 30 s per cycle in a refrigerated chamber (4 &#176;C) for 5 min. Centrifuge at 5,000 x g for 30 s at 4 &#176;C,wait for 1 min on ice to allow the beads to level off, and discard the supernatant.</li>
	<li>Repeat step 6.3 5-10 times.</li>
	<li>Add 10 &#181;L of sample buffer to the sample prepared in step 6.4. Boil at 98 &#176;C for 10 min.</li>
	<li>Centrifuge at 5,000 x g at 4 &#176;C for 30 s.</li>
	<li>Place a column in a new tube with low protein adsorption and transfer the gel to the column using a pipette with a cut tip. A column is used to avoid contamination of the gel.</li>
	<li>Centrifuge at 9,730 x g for 1 min at 4 &#176;C. Collect the flow-through. The sample can be stored at -80 &#176;C. 
	NOTE: If sample degradation is a concern, the following immunoblotting should be performed without freezing.</li>
</ol>
7. Immunoblotting
NOTE: Immunoblotting procedures are based on previous reports7,8.
<ol>
	<li>Load whole samples on a sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE, see Table of Materials). 
	NOTE: If necessary, use large-well gels such that the entire sample can be transferred. Gels with 50 &#181;L wells were used here.</li>
	<li>Set the gels in the electrophoresis chamber and add 1x Tris/Glycine/SDS buffer (see Table of Materials) up to the appropriate volume around the gels.</li>
	<li>Electrophorese gels at 100 V and 400 mA.</li>
	<li>Transfer onto polyvinylidene fluoride (PVDF, see Table of Materials) membranes.</li>
	<li>Block the PVDF membrane blots with 5% skim milk in PBS-P for 1 h at room temperature.</li>
	<li>Wash the membrane thrice with PBS-P on a shaker at top speed for 5 min. Perform the same procedure thereafter when washing.</li>
	<li>Incubate overnight on a shaker with the primary antibody (see Table of Materials) at 4 &#176;C.</li>
	<li>Wash the membrane thrice with PBS-P the next day.</li>
	<li>Incubate for 1 h with the secondary antibody on a shaker at room temperature. 
	NOTE: If the molecular weight of the target protein is close to the IgG heavy chain, which has a molecular weight of about 50 kDa, a light chain-specific secondary antibody can be used to avoid overlap of the target band with the IgG heavy chain. This study used light-chain-specific antibodies7 or Veriblot as IP detection reagent (see Table of Materials).</li>
	<li>Identify bands of proteins with peroxidase luminescent substrates. The membrane can be saved in PBS after washing it twice with PBS-P.</li>
	<li>Strip for 10 min with a stripping solution (see Table of Materials).</li>
	<li>Wash thrice and block with 5% skim milk in PBS-P. The protocol thereafter is the same as in step 7.6 onwards.</li>
</ol>

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

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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. 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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. 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We declare that none of the authors have any conflicts of interest related to this study.

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><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 &amp;micro;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&amp;nbsp;</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>&amp;beta;-Actin (13E5) Rabbit mAb&amp;nbsp;#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.

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

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

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.

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 ...

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897 Views.  Hiroshima University. 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.

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

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Biochemistry

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www.jove.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

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On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies

Source: Obama, T. et al., <a href="https://app.jove.com/v/59733">Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique.</a>&#160;J. Vis. Exp.&#160; (2019)
This video demonstrates co-immunoprecipitated protein complexes on a PVDF membrane for protein-protein interaction analysis. The reduced proteins from complexes were treated with trypsin to cleave the individual proteins at the smaller peptides, then, the remaining peptides were extracted from the PVDF membrane. The pulled peptide derived from both proteins in the complex was then dried and resuspended in a low concentration of formic acid for further analysis.

Source: Obama, T. et al., Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique.&#160;J. Vis. Exp.&#160; (2019)
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Protein-protein interactions

Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells

Source: Strazza, M., et al. <a href="https://app.jove.com/v/58433">Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes.</a> J. Vis. Exp. (2018).
In this video, we demonstrate a technique to perform the co-immunoprecipitation of GFP-tagged wild-type and mutated PD-1 proteins using beads coated with anti-GFP antibodies. The bead-bound PD-1-GFP proteins are incubated with SHP2 proteins that interact with PD-1, enabling the identification of specific structural motifs in PD-1 that play a critical role in SHP2 interaction.

Source: Strazza, M., et al. Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes. J. Vis. Exp. (2018).
In this ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...