Name: tandem affinity purification of protein complexes from eukaryotic cells
Uploaded: 2017-01-26T14:00:00
Description: Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under award number R01AI114362 and Welch Foundation grant I-1782 to Iv&#225;n D'Orso.

1. Plating Cells
<ol>
	<li>One day prior to transfection, plate 3.5 x 106 HEK293T cells in 10 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/Streptomycin (10,000 U/ml stock) per 100 mm dish. Plate 3 - 5 100 mm dishes per experiment/condition to ensure sufficient protein recovery. 
	&#8203;NOTE: The number of plates has to be determined for each experiment/condition, which will rely on the expression levels and solubility of ...

<ol>
	<li>Sharan, R., Ulitsky, I., Shamir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network-based+prediction+of+protein+function.">Network-based prediction of protein function.</a> Mol Syst Biol. 3, 88-99 (2007).</li><li>Jager, 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=Global+landscape+of+HIV-human+protein+complexes.">Global landscape of HIV-human protein complexes.</a> Nature. 481, 365-370 (2012).</li><li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340, 245-246 (1989).</li><li>Luo, Y., Batalao, A., Zhou, H., Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+two-hybrid+system:+a+complementary+approach+to+the+yeast+two-hybrid+system.">Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system.</a> Biotechniques. 22, 350-352 (1997).</li><li>Lee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coimmunoprecipitation+assay.">Coimmunoprecipitation assay.</a> Methods Mol Biol. 362, 401-406 (2007).</li><li>Puig, O., 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=The+tandem+affinity+purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 (2001).</li><li>Jager, 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=Purification+and+characterization+of+HIV-human+protein+complexes.">Purification and characterization of HIV-human protein complexes.</a> Methods. 53, 13-19 (2011).</li><li>Price, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P-TEFb,+a+cyclin-dependent+kinase+controlling+elongation+by+RNA+polymerase+II.">P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II.</a> Mol Cell Biol. 20, 2629-2634 (2000).</li><li>Peterlin, B. M., Price, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+elongation+phase+of+transcription+with+P-TEFb.">Controlling the elongation phase of transcription with P-TEFb.</a> Mol Cell. 23, 297-305 (2006).</li><li>McNamara, R. P., McCann, J. L., Gudipaty, S. A., D'Orso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+mediate+the+enzymatic+disassembly+of+promoter-bound+7SK+snRNP+to+locally+recruit+P-TEFb+for+transcription+elongation.">Transcription factors mediate the enzymatic disassembly of promoter-bound 7SK snRNP to locally recruit P-TEFb for transcription elongation.</a> Cell Rep. 5, 1256-1268 (2013).</li><li>Adelman, K., Lis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter-proximal+pausing+of+RNA+polymerase+II:+emerging+roles+in+metazoans.">Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans.</a> Nat Rev Genet. 13, 720-731 (2012).</li><li>D'Orso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=7SKiing+on+chromatin:+move+globally,+act+locally.">7SKiing on chromatin: move globally, act locally.</a> RNA Biol. 13, 545-553 (2016).</li><li>McNamara, R. P., Bacon, C. W., D'Orso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+Elongation+Control+by+the+7SK+snRNP+Complex:+Releasing+the+Pause.">Transcription Elongation Control by the 7SK snRNP Complex: Releasing the Pause.</a> Cell Cycle. 15, 2115-2123 (2016).</li><li>Yao, F., Svensjo, T., Winkler, T., Lu, M., Eriksson, C., Eriksson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracycline+repressor,+tetR,+rather+than+the+tetR-mammalian+cell+transcription+factor+fusion+derivatives,+regulates+inducible+gene+expression+in+mammalian+cells.">Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells.</a> Hum Gene Ther. 9, 1939-1950 (1998).</li><li>McNamara, R. 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=KAP1+Recruitment+of+the+7SK+snRNP+Complex+to+Promoters+Enables+Transcription+Elongation+by+RNA+Polymerase+II.">KAP1 Recruitment of the 7SK snRNP Complex to Promoters Enables Transcription Elongation by RNA Polymerase II.</a> Mol Cell. 61, 39-53 (2016).</li><li>Ausubel, F. M., 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> Current Protocols in Molecular Biology. , (1994).</li><li>Trudgian, D. C., 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=Comparative+evaluation+of+label-free+SINQ+normalized+spectral+index+quantitation+in+the+central+proteomics+facilities+pipeline.">Comparative evaluation of label-free SINQ normalized spectral index quantitation in the central proteomics facilities pipeline.</a> Proteomics. 11, 2790-2797 (2011).</li><li>Choudhary, C., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+signalling+networks+by+mass+spectrometry-based+proteomics.">Decoding signalling networks by mass spectrometry-based proteomics.</a> Nat Rev Mol Cell Biol. 11, 427-439 (2010).</li><li>Bensimon, A., Heck, A. J., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+proteomics+and+network+biology.">Mass spectrometry-based proteomics and network biology.</a> Annu Rev Biochem. 81, 379-405 (2012).</li><li>Rogers, S., Wells, R., Rechsteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+sequences+common+to+rapidly+degraded+proteins:+the+PEST+hypothesis.">Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis.</a> Science. 234, 364-368 (1986).</li></ol>

The protocol described here for the expression and isolation of protein complexes from eukaryotic cells is not limited to the biochemical characterization of such molecular assemblies, but can also be utilized for the identification of novel interactors and post-translational modifications that could regulate their function. The utilization of affinity tags is not restricted to what is mentioned in this protocol; but our experience suggests that using STREP as the first AP step significantly enhances the yield of the fin...

Protein-protein interactions (PPIs) are critical for the precise regulation of biological processes1, and further studies on these PPIs can inform about their function2. Several approaches have been devised for the study and characterization of PPIs as well as for the de novo identification of novel regulatory protein components. In 1989, Stanley Fields and colleagues reported the yeast two-hybrid (Y2H) assay3. This approach allows for the unbiased and comprehensive identification of interactors (preys) for a defined protein of interest (bait) in Saccharomyces cerevisiae. In addition to its remarkable utility for discovering PPIs, the Y2H assay can be used for characterizing protein pairs in yeast cells, defining minimal interacting domains, and identifying mutations that abolish such interactions. By modifying the Y2H assay, PPIs can also be studied in mammalian cells4. Variations of the Y2H assay (e.g., yeast three-hybrid system) can also be applied to study protein-RNA and protein-small organic ligand interactions in cells.
Another commonly used tool to study PPIs in a homologous system is the co-immunoprecipitation (co-IP) assay5. By using an antibody to immunoprecipitate a protein of interest, the co-IP assay allows researchers to monitor PPIs in cells for various environmental conditions and experimental situations. The use of epitope-tagged proteins (e.g., FLAG, Myc, STREP, and HA, among others) in affinity purification (AP) methods has facilitated the isolation of proteins from complex protein mixtures for several downstream assays, including western blot, silver stain, and enzymatic analysis. However, none of these previous approaches enable the isolation of large quantities of protein complexes for further characterization including in vitro assays, discovery of regulatory subunits by mass spectrometry, and identification of post-translational modifications. An improved version of the AP method is called Tandem AP (TAP), which is a purification technique for studying PPIs by creating a fusion protein with two epitopes that is purified through two subsequent APs6,7. In this article, we present a variation of the TAP method for purifying protein complexes in which two subunits are tagged with different epitopes and then purified through two sequential APs (STREP AP followed by FLAG IP). We first provide a minimalistic overview of TAP (Figure 1) and then a detailed description of all the experimental steps (Figure 2), so that researchers can apply them to their protein complex of interest.
To demonstrate the applicability of the TAP method, we chose a well-characterized cyclin-CDK complex (referred to as P-TEFb kinase), which is composed of the regulatory subunit cyclin T1 (CycT1) and a kinase (CDK9), and is involved in the regulation of transcription by RNA polymerase II (Pol II)8,9,10. P-TEFb phosphorylates the C-terminal domain of Pol II and its associated negative elongation factors, which relieves transcriptional pausing at the promoter and thereby facilitates transcription elongation11,12,13. With this known interaction in mind, STREP-tagged CycT1 and FLAG-tagged CDK9 were over expressed in HEK293T cells. A reciprocal TAP experiment was performed with STREP-tagged CDK9 and FLAG-tagged CycT1 to further validate that the protein interaction is independent of the epitopes utilized. Cells were collected and lysed 48 h post transfection. The soluble lysate was purified by TAP (STREP AP followed by FLAG IP). Input and purified proteins were analyzed by western blot and silver stain (Figure 3).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Fisher Scientific</td>
			<td>SH30022FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30071</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>MP091670049</td>
			<td></td>
		</tr>
		<tr>
			<td>PolyJet</td>
			<td>Fisher Scientific</td>
			<td>50-478-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail&#160;</td>
			<td>Roche&#160;</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>STREP-Tactin Superflow</td>
			<td>IBA</td>
			<td>2-1208-010</td>
			<td></td>
		</tr>
		<tr>
			<td>STREP-tag elution buffer</td>
			<td>IBA</td>
			<td>2-1000-025</td>
			<td></td>
		</tr>
		<tr>
			<td>EZview Red ANTI-FLAG M2 Affinity Gel</td>
			<td>Sigma</td>
			<td>SLBQ1981V</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 100mm &#215; 20 mm style Dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td>
			<td>Corning&#160;</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Lo-Bind Eppendorf</td>
			<td>Fisher Scientific</td>
			<td>13-698-794</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Vortex Mixer</td>
			<td>Fisher Scientific&#160;</td>
			<td>02-215-370</td>
			<td></td>
		</tr>
		<tr>
			<td>48 hole micro tube foam rack</td>
			<td>Fisher Scientific</td>
			<td>02-215-386</td>
			<td></td>
		</tr>
		<tr>
			<td>Labquake shaker rotisserie&#160;</td>
			<td>Thermo&#160;</td>
			<td>415110</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this article, we demonstrate the applicability of the TAP method to the well-characterized CycT1-CDK9 complex (also known as P-TEFb kinase).
Plasmids encoding Cyclin T1-STREP (CycT1:S) and CDK9-FLAG (CDK9:F), or CDK9-STREP (CDK9:S) and Cyclin T1-FLAG (CycT1:F) (Table 1), were transfected into HEK293T cells. Negative controls included transfections with an empty vector and the CDK9:F or CycT1:F plasmids (<stro...

Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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 ...

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