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Identification of Components of RNAi Pathways
Using the Tandem Affinity Purification Method
Mikiko C. Siomi and Haruhiko Siomi
1. Introduction
RNA interference (RNAi) is rapidly becoming a standard laboratory techniquefor understanding and regulating the function of specific genes in evolutionarily
diverse organisms, including plants, Caenorhabditis elegans, Drosophila, andmammalian cells (1–10). RNAi is initiated by the conversion of doublestranded RNA (dsRNA) into 21- to 23-nucleotide (nt) fragments of dsRNA by
Dicer enzymes. These short, interfering RNAs, or siRNAs as they are known,
are incorporated into an RNAi effector complex, the RNA-induced silencing
complex (RISC), which uses them as guides to target and destroy complementary messenger RNA (mRNA). Recent findings point to a tight connection
between microRNA (miRNA) and RNAi molecular machineries. Recent study
also has led to the unmasking of a widespread biological regulatory mechanism
involving miRNAs (11–17), and there is a wide agreement that the core RNAi
machinery carries out numerous cellular functions by an endogenous pathway
important for normal development in many organisms, including gene regulation, virus resistance, and chromatin remodeling (1–17). Although some components of the RNAi cellular machinery have been identified, the overall picture
is far from clear. We describe the tandem affinity purification (TAP) method(18–20) to isolate protein components of RISC in cultured Drosophila
Schneider-2 (S2) cells. This purification method has allowed us to identify
several RISC components, including Argonaute 2 (AGO2), the Drosophila
homolog of fragile X mental retardation protein (dFMR1), and a DEAD-box
RNA helicase Dmp68 (21,22). Identification of components of RNAi/miRNA
pathways by the TAP method eliminates the need for large-scale sample
From: Methods in Molecular Biology, vol. 309: RNA Silencing: Methods and Protocols
Edited by: G. G. Carmichael © Humana Press Inc., Totowa, NJ

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preparation and overcomes, for example, the potential hazards associated withthe use of a radioisotope, thereby placing the method within the scope of theaverage laboratory.
The expression of TAP-tagged AGO2, which is an essential component for asiRNA-directed RNAi response (22,23), will be used to illustrate the method
utilized for isolation of components of RNAi. This method will be useful forstudying many aspects of RNAi/miRNA machineries.
2. Materials
1. Drosophila Schneider-2 (S2) cells.
2. pRmHa-C-FLAG-His expression vector.
3. pRmHa-TAP expression vector.
4. Schneider’s Drosophila medium.
5. Spinner bottles.
6. CELLFECTIN.
7. pCoBlast.
8. Blastcidin S Hydrochloride.
9. TAP buffer: 10 mM Tris-HCl (pH 8.0); 150 mM NaCl; 0.5% Triton X-100 (can be
replaced by 0.05–0.1% NP-40); protease inhibitors.
10. IgG Sepharose.
11. TEV protease.
12. TEV buffer: 10X Stock buffer and ditheithreitol (DTT) solution are supplied with
TEV enzyme when purchased from Invitrogen. Prepare 1X TEV buffer from thestock solution according to the manufacturer’s instruction.
13. Calmodulin affinity resin.
14. Calmodulin-binding buffer: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton
X-100; 10 mM β-Mercaptoethanol, 1 mM MgOAc, 1 mM Imidazole, 2 mM CaCl2.
15. Calmodulin elution buffer: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton
X-100; 10 mM β-Mercaptoethanol; 1 mM MgOAc, 1 mM Imidazole, 2 mM EGTA
(the concentration of EGTA can be higher (up to 10 mM) if the elution efficiency
is lower than desired).
16. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) equipment.
17. Coomassie brilliant blue staining solution: 0.22% Coomassie blue R; 50%
Methanol; 10% Acetic acid.
18. SYPRO Ruby (Molecular Probes, Inc.). SYPRO Ruby staining is done according
to the manufacture’s manual (see Subheading 3.4.).
19. Silver staining kit (Wako, Inc.). Silver staining is done according to the manufacturer’s manual (see Subheading 3.4.).

3. Methods
The methods described below outline (1) the construction of the TAP-tagged
expression plasmid, (2) the transfection of the plasmid DNA and the inductionof protein expression, (3) the purification of the TAP-tagged protein and its

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associated proteins from S2 cells, and (4) the identification of associated proteins by mass spectrometric peptide sequencing.
3.1. Expression Plasmid
The pRmHa-C expression system was a kind gift of Dr. Frank Lafont (24)and is based on the pRmHa-3 (25). pRmHa-3 contains the Drosophila metallothionein promoter, which is activated by Cu2+ and a polyadenylation signal.
pRmHa-3 was modified by introducing a multiple cloning site (EcoRI-SacINheI-KpnI-SmaI-BamHI-Flag tag-EcoRV-10 × His-SalI), FLAG-tag, and His ×
10 tag downstream of the metallothionein promoter to generate pRmHa-C(25). The pBS1479 plasmid was a kind gift of Dr. Bertrand Seraphin (18,19)
and is a yeast expression vector that contains a TAP tag consisting of two
IgG-binding domains of Staphylococcus aureus protein A and a calmodulinbinding peptide (CBP) separated by a TEV protease cleavage site. A BamHIHindIII fragment containing the TAP tag of pBS1479 was subcloned into
pBluScript SK (Stratagene, Inc.), which had been cut with BamHI and HindIII.
A recombinant plasmid was isolated and designated pBlue-TAP. A BamHISalI fragment of pBlue-TAP was subcloned into pRmHa-C that had beendigested with BamHI and SalI, and the desired plasmid was designated
pRmHa-TAP. A cDNA encoding a protein of interest (in this case, AGO2) was
subcloned into a multiple cloning site (EcoRI-SacI-NheI-KpnI-SmaI-BamHI)of the plasmid (see Note 1), which gave rise to a C-terminal TAP tag fusion
gene (see Fig. 1).
3.2. Protein Induction
The next steps in this process involve the transfection of the Schneider line 2(S2) of Drosophila with the expression plasmid for TAP-tagged AGO2
followed by induction with CuSO4 to achieve the production of the fusion
protein.
3.2.1. S2 Cells
The S2 line is a highly versatile expression system, useful for both theanalysis of gene function and generating substantial quantities of expressed
protein (25,26). It was originally derived from primary cultures of late-stage
Drosophila melanogaster male embryos. S2 cells have also been demonstrated to be amenable to RNAi, thereby permitting selective silencing ofgene activity.
The S2 cells were grown at room temperature (25°C) under normal atmosphere in Schneider’s Drosophila medium (Gibco, Inc.) supplemented with 10%
fetal calf serum (FCS) at densities between 5 × 105 and 5 × 106 cells/mL. Mass
cultures were grown in 1-L spinner bottles in 400 mL of medium.

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Fig. 1. Schematic drawing of pRmHa-AGO2-TAP. The TAP is tagged at the Cterminus of AGO2. CBP: calmodulin-binding peptide; TEV: TEV protease cleavage
site; ProtA: two IgG binding domains of Staphylococcus aureus protein A.

3.2.2. Transfection
Transfection was performed using CELLFECTIN (Life Technologies, Inc.)
as follows:
1. Put 250 µL of FCS-free medium in each of two 1.7-mL Eppendorf tubes.
Tube 1: Add 2–6 µg of plasmid DNA into the tube and mix well by pipetting. If
wishing to make a stable line, add 0.1–0.3 µg of a plasmid containing cDNAof a blasticidin-resistant gene (pCoBlast; Invitrogen, Inc.) additionally.
Tube 2: Add 8 µL of CELLFECTIN into the tube and mix well by pipetting.
2. Add the well-mixed DNA solution into the CELLFECTIN solution tube and mix
well by pipetting.
3. Incubate at room temperature (RT) for 20 min.
4. During the incubation time, wash S2 cells (about 1 × 107 cells) with FCS-free
medium one or two times.
5. Resuspend the cells with 0.8 mL of FCS-free medium in a 15-mL conical tube.

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56. Add the DNA/CELLFECTIN solution into the cell suspension and mix by gentle
tapping.
7. Incubate the mixture for 3 h at 25°C.
8. After 3 h incubation, spin down the cells at 400–700g for 5 min. Discard thesupernatant and resuspend the cells with 8 mL of S2 medium containing 10% FCS
and incubate at 25°C.
9. For heterologous protein expression, on d 2, one-half of the transfected cells are
incubated with CuSO4 at 1 mM, and cells are harvested after 12 h.
10. To generate stably transfected S2 cell lines, on the same day, add blasticidin(Blastcidin S Hydrochloride; Waken, Inc.) at 25 µg/mL to the other half of thetransfected cells. After 2–3 wk of selection, resistant clones have grown. Cells are
replated in fresh medium every 4 d over the selection period. These clones are kept
together and are used as polyclonal cell lines. Note that it is difficult to obtain
monoclonal S2 cell lines mainly because S2 cells grow poorly at low densities.
Frozen stocks are kept in 80% FCS, 10% dimethyl sulfoxide at –80°C.

3.3. TAP Purification
Protein purification by the TAP method is performed as follows.
1. Harvest S2 cells expressing the TAP-tagged protein of interest.
2. Wash the cells twice with cold PBS.
3. Resuspend the cells in cold TAP buffer. One milliliter of TAP buffer is needed per
107–108 cells.
4. Pass the cell suspension through a 25-gauge needle attached to a syringe fivetimes. During this step, the sample should be kept on ice. If cells are not disrupted
enough at this point, pass the sample several times through a 30-gauge needle after
the 25-gauge needle passage to ensure cell lysis.
5. Transfer the sample to 1.7-mL microtubes and spin at 16,000g for 1–2 min at 4°C.
6. Take the supernatant and transfer it to new microtubes. This is the cytoplasmic
lysate used for further purification steps. If you wish to obtain the whole-celllysate at this point, the cell suspension at step 4 should be sonicated on ice andsteps 5 and 6 then followed.
7. The supernatant is now mixed with IgG SepharoseTM 6 Fast Flow beads(Amersham Biosciences, Inc.) in microtubes and rocked for 1–2 h at 4°C. Prior tothis step, an aliquot of the IgG beads (approx 30–40 µL) is placed into a 1.7-mL
microtube, washed with TAP buffer several times, and kept on ice.
8. Wash the IgG beads with TAP buffer five times (see Note 2). After the last wash,
spin the tubes briefly to collect residual buffers to the bottom, suck them up, anddiscard.
9. Wash with TEV buffer once. Drain the beads well, as in step 8. Add TEV buffer
to the beads. The buffer volume used here is about 500 µL per tube.
10. Add TEV (Invitrogen) to the beads. The enzyme volume added is 1/100 vol of thebuffer. Rock at 16°C for 2 h.
11. Spin at 16,000g for 1–2 min at 4°C. Transfer the supernatant into new microtubes.

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Fig. 2. Purification of proteins that associate with AGO2 by using a C-terminal TAP
tag. The protein components in the “IgG bound” obtained from S2 cells expressing the Cterminal TAP-tagged AGO2 (AGO2-TAP) and the parental cells (control) were resolved
on SDS-PAGE and visualized by Coomassie blue staining. Several distinct bands (indicated with asterisks) are observed only in the AGO2–TAP lane. The purification was done
from 150 mL of S2 cell culture. The left lane represents molecular markers.
12. Add 100 µL of TEV buffer to the beads, and mix well by tapping. Take the supernatant after the spinning at 16,000g for 1–2 min at 4°C and combine it with thesample obtained at step 11.
13. Spin again at 16,000g for 1–2 min at 4°C to make sure all of the beads sedimentat the bottom of the tubes. Take the supernatant and transfer it to new tubes.
14. Add 3 mL of calmodulin-binding buffer and 2 µL of 1 M CaCl2 per 1 mL of thesample.
15. Mix with calmodulin affinity resin (Stratagene, Inc.) and rock for 1 h at 4°C. An
aliquot of the calmodulin beads (approx 30–40 µL) is placed in each microtube
and washed with calmodulin-binding buffer several times prior to use.
16. Wash the beads with calmodulin-binding buffer five times. After the last wash,
spin briefly to collect the residual buffer to the bottom and drain well.

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17. Add EGTA-containing elution buffer to the beads; rock at RT for 15 min or
longer to elute the TAP complexes. If the volume is higher than desired at thispoint, perform a trichloroacetic acid (TCA) precipitation to concentrate theprotein sample.
18. Run the sample on a protein gel and stain to visualize the protein bands.

3.4. Identification of TAP Affinity Purified Proteins
The final extracts were visualized on SDS-PAGE gels by Coomassie brilliant
blue (ICN Biomedicals, Inc.) staining, SYPRO Ruby staining using a SYPRO
Ruby Protein gel stain kit (Molecular Probes, Inc.), or silver staining using
SilverQuest Silver Staining kit (Invitrogen) or Silver Stain II kit (Wako, Inc).
Stained bands (see Fig. 2) were excised from the gels, digested with trypsin,and processed for mass spectrometric fingerprinting as described (21). Using
mass spectrometry and the complete Drosophila genome (the Celera/Berkeley
Drosophila protein database), it is relatively easy to identify each of the specificbands on the gels.
4. Notes
1. Usually, standard DNA cloning procedures can be used to introduce the C-terminal
TAP tag in frame with the coding region of the protein of interest. The upstream
sequence of the initiation ATG is often very important for getting sufficient
expression levels of the TAP-tagged protein of interest. If the Kozak sequence ofthe gene of interest is not close to the consensus Kozak sequence of Drosophilamelanogaster, you might need to modify the sequence upstream (and downstream) of the initiation ATG of the gene.
2. If the background is high when you visualize proteins recovered following TAP
purification, you might use a higher salt concentration in the washing buffer.
However, of course, a high salt level would disrupt the interaction of TAP-tagged
proteins with proteins. Therefore, you might have to selectively adjust TAP purification conditions (salt and/or detergent concentrations, incubation time for binding, and so on).

Acknowledgments
The authors thank Dr. B. Seraphin for providing the TAP plasmids. We also
thank Dr. F. Lafont for the pRmHa plasmid. This work was supported by grants
from the Ministry of Education, Culture, Sports, Science, and Technology ofJapan (MEXT) and the Japan Society for the Promotion of Science (JSPS).
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