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The tandem affinity purification (TAP) procedure was pioneered in yeast for the purpose of purifying and characterizing protein complexes. While affinity purification is relatively easy to perform, nonspecific protein interactions can plague the identification of true interacting partners of the given bait utilized in the purification. To alleviate this problem, two sequential affinity purification steps are employed in the TAP procedure. Since its inception in yeast, TAP has gone through many adaptations and has been employed multiple times in diverse organisms, including mammalian systems. In all these approaches, two out of many possible affinity moieties are employed and are usually expressed as a fusion polypeptide in the amino or carboxyl-terminal region of the protein bait. In this protocol, we describe a variation on the TAP procedure in which the affinity moieties are placed on two different proteins of a molecular complex to isolate or detect components present in the complex. This variation, which we refer to as bimolecular affinity purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known components.
This procedure is a variation of the conventional TAP procedure (Figure 1). The protocol will focus on two affinity moieties, glutathione s-transferase (GST) and a biotinylation peptide from transcarboxylase. The protocol was utilized to isolate a protein complex containing both GCN5 and COMMD1 and confirm that this complex also interacts with the NF-κB subunit RelA (Mao 2009).
A method similar to the calcium phosphate precipitation in Step 3 can be found in Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs (Sambrook and Russell 2006).
Bait DNA sequences
BAP wash buffer
Biotin, 4 mM (Cell-culture tested)
CaCl2, 2 M (Cell-culture tested)
Cell culture medium
For HEK293 cells, we utilize Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine.
GST fusions: pEBG or pEBB-C-GST (or an equivalent expression system)
Biotinylation target peptide: pEBB-C-TB (or an equivalent expression system)
Glutathione elution buffer for BAP
Glutathione resin (immobilized onto agarose or Sepharose beads)
HBSS for BAP, 2X
L-Glutathione, reduced (>98% purity)
PBS (phosphate-buffered saline), 1X, cold
Reagents and equipment for western blotting (see Step 13)
Triton lysis buffer for BAP
“Complete” Triton lysis buffer is made by adding protease inhibitors, as described in the recipe.
Strepavidin resin (immobilized onto agarose or Sepharose beads)
Cell scraper, 3-cm blade
Centrifuge chromatography columns, 22-mL total volume (Pierce, 89898)
Benchtop centrifuge with cooling system for 1.5-ml microcentrifuge tubes
Tabletop centrifuge with a fixed-angle rotor that can reach 15,000g (for example Eppendorf 5810R centrifuge and F34-6-38 rotor).
Conical tubes, 50-mL, able to withstand 15,000g (Corning, 430828)
Laminar flow hood and CO2 incubators for cell culture
Microcentrifuge tubes, 1.5-ml
Serological pipets and pipet-aid
Tissue culture plates, 15-cm
A quality-control western blot should be performed to ensure that proper precipitation and elution of each one of the baits has occurred.
Problem: Bait 1 did not elute from the GSH column.
Solution: The elution of Bait 1 depends on the presence in the elution buffer of glutathione and a mild detergent (0.1% Triton X-100 in this case). A single elution step has variable efficiency, and sequential elution steps as described here are advisable for most baits, although this will dilute the final eluate. In addition to employing sequential elutions, make sure that the glutathione has been stored properly (at 4°C, with desiccation).
Problem: Bait 2 was not precipitated in the final purification.
Solution: There are at least two possible explanations: (1) Bait 2 did not co-precipitate with Bait 1 in the initial GSH precipitation. This should be evident when looking at the sample from the GSH column prior to elution as well as in the GSH eluate. The BAP procedure presumes that the ability of both baits to interact is well established. However, the affinity tags and their positioning may impair the interaction between two proteins. If the tags are thought to be affecting the interaction between the baits, there are two potential steps that can be taken. Either the GST tag can be moved to the opposite end of Bait 1, or other elutable affinity tags such as tandem epitope tags can be utilized instead of GST (Table 1). (2) Precipitation of Bait 2 may be impaired if the fusion protein is not properly biotinylated in vivo. While Bait 2 may be expressed well, in some cases biotinylation of the transcarboxylase peptide is deficient in certain fusion proteins, particularly when placed in the amino terminus (thus we recommend using this peptide always as a carboxyl-terminal fusion). To confirm proper biotinylation of Bait 2, compare immunoblots performed with a protein-specific antibody and with streptavidin-HRP (if the protein is expressed but poorly biotinylated, the signal from streptavidin-HRP will be dramatically lower).
Advances in affinity-based protein purification techniques have resulted in relatively simple purification protocols that are generally accessible to molecular biology laboratories. A commonly utilized format is the TAP procedure, in which protein purification and mass spectrometry analysis are coupled to identify novel protein complexes (Burckstummer 2006; Puig 2001; Rigaut 1999). However, the purification of a given protein bait by this procedure does not mean that a single homogenous molecular complex has been isolated, and such consideration may be critical in certain circumstances. The protocol presented here is intended for the purification of protein complexes marked by the presence of two components and therefore can be tailored to represent a homogenous population (Figure 1). In addition, the BAP procedure is simple and accessible to laboratories that are unable to do more complicated chromatographic separation techniques, but for whom the homogeneity of the sample is important. BAP requires the concurrent expression of two distinct baits and therefore its efficiency may be dramatically affected by differences in relative expression levels of the baits. Similarly, the tags fused to the proteins might alter the complex composition, enzyme activity, or cell homeostasis. Therefore, it is of major importance that proper functionality of the baits is confirmed in the actual expression system prior to the purification. In this regard, while the protocol presented here is based on the GST and TB tags, a number of other affinity moieties could be potentially used with the appropriate modifications in the binding and elution conditions required (Table 1). Given the very high affinity of biotin and streptavidin, the TB tag is an excellent choice for the second purification step in this protocol. This procedure allows for simple confirmation of ternary complexes between the two baits and a potential third interacting partner, as was the case in our study of the GCN5-COMMD1 complex and its interaction with the NF-κB subunit, RelA (Mao 2009). However, depending on the relative stoichiometry of the interaction between the two baits in cells and the scale of the purification performed, significant amounts of the purified complex can be isolated that will be suitable for MS-based identification of novel interaction partners.
BAP wash buffer: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 ng/mL Leupeptin, 1 ng/mL Aprotinin.
Glutathione elution buffer for BAP: 40 mM Glutathione, 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1% Triton X-100, 10 ng/mL Leupeptin, 1 ng/mL Aprotinin.
HBSS for BAP, 2 ×: 50 mM HEPES, 1.5 mM Na2HPO4, 280 mM NaCl. To prepare this buffer, we start from the solid forms of each chemical and dissolve them in 400 mL of ultra pure water, using a sterile glass beaker. Adjust the pH to 7.00 (using NaOH or HCl as needed), and then bring the volume up to 500 mL with additional ultra pure water. Adjust pH again and sterilize by filtration in a laminar flow hood (0.2 μm pore filter bottles, Corning 430769). Test the batch for transfection efficiency using a reporter plasmid (expressing EGFP for example). If the transfection efficiency is adequate, this reagent can be stored for many months at 4°C for repeated use.
Triton lysis buffer for BAP: 25 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10% (v/v) Glycerol, 1% (v/v) Triton X-100. This buffer can be made ahead of time and stored at RT. Just prior to use, add the following to make “complete” Triton lysis buffer: 1 mM PMSF, 10 mM DTT, 1 mM Sodium orthovanadate, 10 ng/mL Leupeptin, 1 ng/mL Aprotinin. Complete Triton lysis buffer can be stored at −20°C for 4 weeks.