tandem affinity purification assay to study protein-protein interactions

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

After the cell lysate and STREP-bead slurry mixtures have incubated for 2 hours, spin them down at 1,1500 times g for 2 minutes at 4 degrees Celsius. Save the supernatant as &#34;STREP AP flow-through,&#34; and store at 4 degrees Celsius.
Add 500 microliters of STREP AP Wash Buffer 1 to the beads. Nutate the beads and buffer mixture for 5 minutes at 4 degrees Celsius, and centrifuge at 1,500 times g for 2 minutes at 4 degrees Celsius. Discard the supernatant, and wash again with 500 microliters of STREP AP Wash Buffer 1.
After the third wash, transfer the bead solution to a new 1.5-milliliter microcentrifuge tube to reduce the protein background. Wash a fourth time with STREP AP Wash Buffer 1. After the spin, remove the supernatant, and add 500 microliters of Wash Buffer 2.
Equilibrate the beads by inverting the tubes, and then, spin down at 1,500 times g for 2 minutes at 4 degrees Celsius. Discard the supernatant. Elute the protein of interest with 50 microliters of STREP-elution buffer. Vortex at 800 RPM for 15 minutes at 4 degrees Celsius. Spin down the beads at 1500 times g for 1 minute at 4 degrees Celsius. Collect the supernatant. This fraction corresponds to the &#34;STREP AP elution&#34; sample.
Save the beads fraction at 4 degrees Celsius for a second elution. Aliquot 10% of the STREP AP elution for silver staining and/or western blotting.
To increase the amount of starting material for the subsequent FLAG immunoprecipitation, the first and second STREP elutions can be pooled together for more efficient recovery of the FLAG elution.
Begin this procedure by using a wide-mouthed pipette tip to transfer the appropriate amount of FLAG bead solution to a microcentrifuge tube. Spin down at 1,500 times g for 2 minutes at 4 degrees Celsius. Remove the supernatant, and add 500 microliters of FLAG Wash Buffer to the beads.
Spin down at 1,500 times g for 2 minutes at 4 degrees Celsius. Discard the supernatant, and wash again with FLAG wash buffer. After the third wash, remove the supernatant, and resuspend the beads in FLAG wash buffer to a final volume of n times 16 microliters where n is the number of samples.
Add 16 microliters of the FLAG bead slurry to the remaining STREP AP elution, and nutate overnight at 4 degrees Celsius. Following an overnight incubation of the FLAG bead slurry and STREP AP elution, proceed with FLAG immunoprecipitation.
Spin down the FLAG beads suspension at 1,500 times g for 2 minutes at 4 degrees Celsius. Save the supernatant as the &#34;FLAG IP flow-through,&#34; and store at 4 degrees Celsius.
Add 500 microliters of FLAG wash buffer to the beads. Nutate for 5 minutes at 4 degrees Celsius, and centrifuge at 1,500 times g for 2 minutes at 4 degrees Celsius. Discard the supernatant, and wash again with 500 microliters of FLAG wash buffer.
After the third wash, transfer the protein bead solution to a new 1.5-milliliter microcentrifuge tube to reduce background. Wash a fourth time with FLAG wash buffer. Remove the supernatant from the final spin, and add to the beads 30 microliters of FLAG wash buffer containing 200 nanograms per microliter of FLAG peptide. Vortex at 800 RPM for two hours at 4 degrees Celsius.
After two hours, spin down the suspension at 1,500 times g for 2 minutes at 4 degrees Celsius. Harvest the supernatant, and store at 4 degrees Celsius as the &#34;FLAG IP elution.&#34;

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Biomolecular Interaction Detection Techniques

Protein-protein interactions

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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 mm2 dishes per experiment/condition to ensure sufficient protein recovery. 
	NOTE: The number of plates has to be determined for each experiment/condition, which will rely on the expression levels and solubility of the protein complex of interest. Alternatively, if larger-scale purification is needed, cell lines can be adapted to grow in suspensions in spinner flasks, but this method will not work for transient transfections.</li>
</ol>
2. Transfecting Cells or Inducing Stable Cell Lines
<ol>
	<li>On the following day, check the cells under the microscope. Cells must be 70 - 80% confluent before proceeding.</li>
	<li>Transfect the cells with a total mixture of 7 &#181;g of plasmid DNA and 21 &#181;l of transfection reagent per 100 mm dish. Transfect cells with a vector expressing GFP (Table 1) as a control for transfection efficiency. 
	NOTE: If using an HEK293-T-REx or similar stable cell line that induces the expression of two or more complex subunits in response to doxycycline, the inducer will have to be added to the cells and incubated for 48 hr. Similarly, a stable cell line that induces the expression of GFP upon the addition of the inducer would also be required for validation purposes. Proceed to step 2.7.</li>
	<li>Prepare the transfection mixture per 100 mm dish. In a 1.5 ml microcentrifuge tube, mix 500 &#181;l of unsupplemented DMEM with 7 &#181;g of total plasmid DNA (corresponding to two or more plasmids). In a separate microcentrifuge tube, mix 500 &#181;l of unsupplemented DMEM with 21 &#181;l of the transfection reagent. Repeat for each transfection reaction.</li>
	<li>Pipette the transfection reagent solution into the plasmid DNA solution (do not mix solutions in the reverse order). Mix by pipetting at least 4 times.</li>
	<li>Incubate this mixture at room temperature for 10 - 15 min, but no longer than 15 min.</li>
	<li>Tilt the plate to create a media reservoir and carefully pipette the transfection mixture drop-wise onto the interior side of the plate without disturbing the cells. Return the dish to its original flat position and swirl gently to distribute the mixture.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified cell culture incubator with 5% CO2 (this condition is hereafter referred to as &#34;tissue culture incubator&#34;). Replace the media 5 hr post-transfection (optional).</li>
</ol>
3. Checking Transfection or Induction Efficiency
<ol>
	<li>At 24 hr post-transfection, visualize the cells under a fluorescent microscope to check for transfection efficiency (or induction of control HEK293T-REx stable cell line expressing GFP). Incubate for another 24 hr.</li>
</ol>
4. STREP Affinity Purification (STREP AP)
<ol>
	<li>Collecting the cells
	<ol>
		<li>At 48 hr post-transfection, remove the media and add 8 ml of cold 1x phosphate-buffered saline (PBS). Pipette up and down to detach cells from the plate.</li>
		<li>Collect and transfer the cell suspension into a 15 mL tube and spin the cells down at 800 x g for 4 min at 4 &#176;C.</li>
		<li>Remove the PBS supernatant. Repeat the spin down (800 x g for 1 min at 4 &#176;C) to eliminate any residual PBS. Store the cell pellet at -80 &#176;C for future use or follow the procedure below to continue protein purification.</li>
	</ol>
	</li>
	<li>Lysing the cells
	<ol>
		<li>Resuspend the cell pellets using 5&#215; the cell pellet volume (~250 &#181;l per plate) of Passive Lysis Buffer (PLB: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, protease inhibitor cocktail tablet, 5% v/v glycerol, and 1.0% NP-40) and transfer the cell suspension to a 1.5 ml microcentrifuge tube.</li>
		<li>Briefly vortex the suspension and nutate for 30 min at 4 &#176;C.</li>
		<li>Spin down the cell suspension at 10,000 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer the soluble lysates into a new 1.5 ml microcentrifuge tube and discard the cell pellets. Save 10% of the final volume of the soluble lysate as &#34;STREP AP Input.&#34; Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Equilibrating the STREP beads 
	NOTE: Use STREP beads for the first AP step! Complexes pulled down with STREP beads recover significantly more protein than those pulled down with FLAG beads.
	<ol>
		<li>Take out (n &#215; 40 &#181;l) + n &#181;l of the 50% STREP bead slurry (n = number of samples) and spin down at 1,500 x g for 2 min at 4 &#176;C. 
		&#8203;NOTE: Use wide-mouthed tips to pipette beads.</li>
		<li>Remove the supernatant and add 500 &#181;l of PLB to the beads. Nutate the beads mixture for 5 min at 4 &#176;C. Spin down at 1,500 x g for 2 min at 4 &#176;C.
		<ol>
			<li>Repeat for a total of 3 washes. Resuspend the beads in PLB to a final volume of n &#215; 40 &#181;l.</li>
		</ol>
		</li>
		<li>Add 40 &#181;l of 50% STREP bead slurry to each cell lysate sample and nutate for 2 hr at 4 &#176;C.</li>
	</ol>
	</li>
	<li>STREP AP
	<ol>
		<li>Spin down the solution at 1,500 x g for 2 min at 4 &#730;C.</li>
		<li>Remove the supernatant and store it as &#34;STREP AP Flow-through (FT)&#34; at 4 &#176;C.</li>
		<li>Add 500 &#181;l of STREP AP Wash Buffer I (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 5% glycerol, and 0.2% NP-40) to the beads. Nutate the beads mixture for 5 min at 4 &#176;C and centrifuge at 1,500 x g for 2 min at 4 &#176;C. Discard the supernatant.
		<ol>
			<li>Repeat for a total of 4 washes. 
			NOTE: Before the fourth wash, transfer the beads solution to a new 1.5 ml microcentrifuge tube to reduce the protein background. This step is essential.</li>
		</ol>
		</li>
		<li>Add 500 &#181;l Wash Buffer II (100 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1 mM EDTA) and equilibrate the beads by inverting the tubes. Spin down at 1,500 x g for 2 min at 4 &#176;C and discard the supernatant.</li>
		<li>Elute the protein of interest with 50 &#181;l of STREP Elution Buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, and 2.5 mM Desthiobiotin). Vortex at 800 rpm for 15 min at 4 &#176;C.</li>
		<li>Spin down the beads at 1,500 x g for 1 min at 4 &#176;C. Collect the supernatant. This fraction corresponds to the &#34;STREP AP Elution&#34; sample.</li>
		<li>Save the beads fraction at 4 &#176;C for a second elution, which could yield up to half of the amount of protein obtained with the first elution. Aliquot 10% of the STREP AP Elution for silver staining and/or western blotting.</li>
	</ol>
	</li>
	<li>Equilibrating the FLAG beads
	<ol>
		<li>Take out (n x 16 &#181;l) + n &#181;l of FLAG beads solution (n = number of samples) and spin down at 1,500 x g for 2 min at 4 &#176;C. 
		&#8203;NOTE: Use wide-mouthed tips to pipette beads.</li>
		<li>Remove the supernatant and add 500 &#181;L of FLAG wash buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.1% NP-40) to the beads. Spin down at 1,500 x g for 2 min at 4 &#176;C. Repeat for a total of 3 washes.</li>
		<li>Resuspend the beads in FLAG wash buffer to a final volume of n x 16 &#181;l.</li>
		<li>Add 16 &#181;l of the FLAG bead slurry to the remaining STREP AP elution and nutate overnight at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
5. FLAG IP
<ol>
	<li>Spin down the FLAG beads suspension at 1,500 x g for 2 min at 4 &#176;C. Save the supernatant and store it as the &#34;FLAG IP Flow-through&#34; (FT) at 4 &#176;C.</li>
	<li>Add 500 &#181;l of FLAG wash buffer to the solution. Nutate for 5 min at 4 &#176;C and centrifuge at 1,500 x g for 2 min at 4 &#176;C. Repeat for a total of 4 washes and remove the supernatant after each wash. 
	NOTE: Before the fourth wash, transfer the protein-bead solution to a new 1.5 ml microcentrifuge tube to reduce the background. This step is essential.</li>
	<li>Remove the supernatant from the final spin and add 30 &#181;L of FLAG wash buffer containing 200 ng/&#181;l of FLAG peptide into the beads. Vortex at 800 rpm for 2 hr at 4 &#176;C.</li>
	<li>Spin down the suspension at 1,500 x g for 2 min at 4 &#176;C. Harvest the supernatant and store at 4 &#176;C as the &#34;FLAG IP Elution.&#34; 
	NOTE: The elution samples can be confirmed by silver stain and/or western blot using standard protocols, and are ready for further protein assays. When setting up a western blot, run all of the following samples: (1) STREP AP Input, (2) STREP AP FT, (3) STREP AP Elution, (4) FLAG IP FT, and (5) FLAG IP Elution. Volumes loaded will have to be determined for each experiment. All collected samples and beads can be stored at 4 &#176;C for short-term storage and at -80 &#176;C for long-term storage. TAP samples could be analyzed by mass spectrometry to verify the identity of their components, to identify novel post-translational modifications (PTMs), and/or to discover any novel protein interaction with the purified complex. For this purpose, after running a coomassie gel or silver stain, bands can be excised from the gel using a clean scalpel. Several excellent reviews that discuss mass spectrometry methods were previously published.</li>
</ol>

<table><tbody><tr><td>Dulbecco&#039;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</td><td>Roche</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 &amp;times; 20 mm style Dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td><td>Corning</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</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</td><td>Thermo</td><td>415110</td><td></td></tr></tbody></table>

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.

Table 1: Plasmids used in this Study. &#160;CycT1&#160;and&#160;CDK9&#160;were ligated into the pcDNA/4TO-STREP and pcDNA/4TO-FLAG plasmid vectors. These constructs were transformed into DH5&#945; competent cells and plated on LB-Ampicillin plates. Colonies were selected, grown, and screened. Successfully ligated clones were verified by Sanger sequencing and the combination of restriction digestion and agarose gel electrophoresis. For CycT1 we used a shorter version (1 - 708) instead of the full-length (1 -...

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

Tandem affinity purification is a sequential purification scheme for isolating protein complexes from cells to study protein-protein interactions.
To begin, take a cell culture expressing epitope-tagged proteins of interest. The proteins form a complex inside the cell. One of the proteins in the complex contains a Strep-tag, while the other is labeled with a FLAG-tag. These are small synthetic peptide sequence-based affinity tags.
Remove the medium, and add a detergent solution to lyse the cells. Centrifuge to remove the cellular debris. Collect the supernatant containing the target protein complex and other cellular proteins.
Add Strep-beads &#8212; agarose beads coated with streptavidin &#8212; and incubate. The Strep-tag of the protein complex binds to streptavidin. Centrifuge to collect the protein complex-bound beads. Wash to remove any contaminating cellular proteins.
Add elution buffer containing desthiobiotin &#8212; a biotin derivative &#8212; which displaces the protein complex by competitively binding to streptavidin. Centrifuge to obtain a supernatant containing the eluted protein complex.
Add FLAG-beads &#8212; agarose beads coated with anti-FLAG antibodies &#8212; and incubate. The FLAG-tag of the protein complex binds to the antibody. Centrifuge to collect the protein complex-bound beads.
Add elution buffer containing free FLAG peptide. The peptide competitively binds to the antibody and displaces the bound protein complex. Centrifuge to elute the purified protein complex.
Assess the presence of both proteins in the elute to confirm the interaction between the proteins of interest.

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Tandem Affinity Purification Assay to Study Protein-Protein Interactions

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Tandem Affinity Purification Assay to Study Protein-Protein Interactions