Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Overview

This video demonstrates tandem affinity purification — 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.

Protocol

1. Plating Cells

1. One day prior to transfection, plate 3.5 x 10⁶ HEK293T cells in 10 mL of Dulbecco'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.

2. Transfecting Cells or Inducing Stable Cell Lines

1. On the following day, check the cells under the microscope. Cells must be 70 - 80% confluent before proceeding.
2. Transfect the cells with a total mixture of 7 µg of plasmid DNA and 21 µl 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.
3. Prepare the transfection mixture per 100 mm dish. In a 1.5 ml microcentrifuge tube, mix 500 µl of unsupplemented DMEM with 7 µg of total plasmid DNA (corresponding to two or more plasmids). In a separate microcentrifuge tube, mix 500 µl of unsupplemented DMEM with 21 µl of the transfection reagent. Repeat for each transfection reaction.
4. 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.
5. Incubate this mixture at room temperature for 10 - 15 min, but no longer than 15 min.
6. 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.
7. Incubate the cells at 37 °C in a humidified cell culture incubator with 5% CO₂ (this condition is hereafter referred to as "tissue culture incubator"). Replace the media 5 hr post-transfection (optional).

3. Checking Transfection or Induction Efficiency

1. 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.

4. STREP Affinity Purification (STREP AP)

1. Collecting the cells
   1. 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.
   2. 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 °C.
   3. Remove the PBS supernatant. Repeat the spin down (800 x g for 1 min at 4 °C) to eliminate any residual PBS. Store the cell pellet at -80 °C for future use or follow the procedure below to continue protein purification.
2. Lysing the cells
   1. Resuspend the cell pellets using 5× the cell pellet volume (~250 µl 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.
   2. Briefly vortex the suspension and nutate for 30 min at 4 °C.
   3. Spin down the cell suspension at 10,000 x g for 10 min at 4 °C.
   4. 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 "STREP AP Input." Store at 4 °C.
3. 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.
   1. Take out (n × 40 µl) + n µl of the 50% STREP bead slurry (n = number of samples) and spin down at 1,500 x g for 2 min at 4 °C.
      ​NOTE: Use wide-mouthed tips to pipette beads.
   2. Remove the supernatant and add 500 µl of PLB to the beads. Nutate the beads mixture for 5 min at 4 °C. Spin down at 1,500 x g for 2 min at 4 °C.
      1. Repeat for a total of 3 washes. Resuspend the beads in PLB to a final volume of n × 40 µl.
   3. Add 40 µl of 50% STREP bead slurry to each cell lysate sample and nutate for 2 hr at 4 °C.
4. STREP AP
   1. Spin down the solution at 1,500 x g for 2 min at 4 ˚C.
   2. Remove the supernatant and store it as "STREP AP Flow-through (FT)" at 4 °C.
   3. Add 500 µl 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 °C and centrifuge at 1,500 x g for 2 min at 4 °C. Discard the supernatant.
      1. 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.
   4. Add 500 µl 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 °C and discard the supernatant.
   5. Elute the protein of interest with 50 µl 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 °C.
   6. Spin down the beads at 1,500 x g for 1 min at 4 °C. Collect the supernatant. This fraction corresponds to the "STREP AP Elution" sample.
   7. Save the beads fraction at 4 °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.
5. Equilibrating the FLAG beads
   1. Take out (n x 16 µl) + n µl of FLAG beads solution (n = number of samples) and spin down at 1,500 x g for 2 min at 4 °C.
      ​NOTE: Use wide-mouthed tips to pipette beads.
   2. Remove the supernatant and add 500 µL 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 °C. Repeat for a total of 3 washes.
   3. Resuspend the beads in FLAG wash buffer to a final volume of n x 16 µl.
   4. Add 16 µl of the FLAG bead slurry to the remaining STREP AP elution and nutate overnight at 4 °C.

5. FLAG IP

1. Spin down the FLAG beads suspension at 1,500 x g for 2 min at 4 °C. Save the supernatant and store it as the "FLAG IP Flow-through" (FT) at 4 °C.
2. Add 500 µl of FLAG wash buffer to the solution. Nutate for 5 min at 4 °C and centrifuge at 1,500 x g for 2 min at 4 °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.
3. Remove the supernatant from the final spin and add 30 µL of FLAG wash buffer containing 200 ng/µl of FLAG peptide into the beads. Vortex at 800 rpm for 2 hr at 4 °C.
4. Spin down the suspension at 1,500 x g for 2 min at 4 °C. Harvest the supernatant and store at 4 °C as the "FLAG IP Elution."
   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 °C for short-term storage and at -80 °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.

Representative Results

Table 1: Plasmids used in this Study.  CycT1 and CDK9 were ligated into the pcDNA/4TO-STREP and pcDNA/4TO-FLAG plasmid vectors. These constructs were transformed into DH5α 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 - 726) because the last 18 residues contain a PEST sequence (a peptide sequence rich in proline, glutamic acid, serine, and threonine), which acts as a signal peptide for protein degradation. CDK9 was full-length. The sequence of the tandem STREP tag is as follows: GGGGWSHPQFEKGGGSGGGSGGGSWSHPQFEK. The sequence of the tandem FLAG tag is as follows: GGGGDYKDHDGDYKDHDIDYKDDDDK.

Protein	Residues	Vector	Tag	Cloning sites	Reference
CycT1	1-708	pcDNA/4TO	STREP	HindIII - EcoRI	McNamara et al., 2013
CycT1	1-708	pcDNA/4TO	FLAG	HindIII - EcoRI	This article
CDK9	1-372	pcDNA/4TO	STREP	HindIII - XhoI	This article
CDK9	1-372	pcDNA/4TO	FLAG	HindIII - XhoI	McNamara et al., 2013
GFP	1-238	pcDNA/4TO	STREP-FLAG	BamHI - XhoI	This article

Materials

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	Fisher Scientific	SH30022FS
Fetal bovine serum	Hyclone	SH30071
Penicillin/Streptomycin	Fisher Scientific	MP091670049
PolyJet	Fisher Scientific	50-478-8
Protease inhibitor cocktail	Roche	11836153001
STREP-Tactin Superflow	IBA	2-1208-010
STREP-tag elution buffer	IBA	2-1000-025
EZview Red ANTI-FLAG M2 Affinity Gel	Sigma	SLBQ1981V
Corning 100mm × 20 mm style Dish cell culture treated nonpyrogenic polystyrene 20/sleeve	Corning	430167
Protein Lo-Bind Eppendorf	Fisher Scientific	13-698-794
Digital Vortex Mixer	Fisher Scientific	02-215-370
48-hole micro tube foam rack	Fisher Scientific	02-215-386
Labquake shaker rotisserie	Thermo	415110