PROTOCOL

Detecting proteinprotein interactions by far western blotting

Yuliang Wu1,2, Qiang Li1 & Xing-Zhen Chen1
1Membrane

Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada. 2Present address: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA. Correspondence should be addressed to X.-Z.C. (xzchen@ualberta.ca). Published online 13 December 2007; doi:10.1038/nprot.2007.459

2007 Nature Publishing Group http://www.nature.com/natureprotocols

Far western blotting (WB) was derived from the standard WB method to detect proteinprotein interactions in vitro. In Far WB, proteins in a cell lysate containing prey proteins are rstly separated by SDS or native PAGE, and transferred to a membrane, as in a standard WB. The proteins in the membrane are then denatured and renatured. The membrane is then blocked and probed, usually with puried bait protein(s). The bait proteins are detected on spots in the membrane where a prey protein is located if the bait proteins and the prey protein together form a complex. Compared with other biochemical binding assays, Far WB allows prey proteins to be endogenously expressed without purication. Unlike most methods using cell lysates (e.g., co-immunoprecipitation (co-IP)) or living cells (e.g., uorescent resonance energy transfer (FRET)), Far WB determines whether two proteins bind to each other directly. Furthermore, in cases where they bind to each other indirectly, Far WB allows the examination of candidate protein(s) that form a complex between them. Typically, 23 d are required to carry out the experiment.

INTRODUCTION The study of proteinprotein interactions is vital to understand how proteins function within a cell. A variety of approaches for detecting proteinprotein interactions have been developed in the past two decades13. Generally, they are divided into in vivo and in vitro approaches. Commonly used in vivo approaches include two-hybrid systems, using bacteria, yeast or mammalian cells as host46; uorescence resonance energy transfer (FRET)7 and bioluminescence resonance energy transfer (BRET)8, using living cells; and the synthetic lethality method in yeast9. There are numerous in vitro approaches, such as tandem afnity purication and mass spectrometry10, surface plasmon resonance (SPR)11, protein microarray12, dot blotting13, co-immunoprecipitation (co-IP) using cultured cells or tissues and pull-down assays using glutathione S-transferase (GST), His or FLAG tags14. A two-hybrid system is based on the activation of reporter gene(s) once two proteins associate with each other. It is mostly applied to soluble proteins (as is GST pull-down) though systems for membrane proteins do exist15; however, a major disadvantage with this technique is the presence of false-positives and false-negatives. FRET and BRET use living cells but require the construction of expression vectors and need specic equipment7,8. In addition, as seen with co-IP, these methods cannot determine whether two proteins directly bind to each other. Both SPR and protein microarray also need specic instruments. Dot blotting is as simple as Far western blotting (WB), but it does not provide the information on the size of binding partner(s). Far WB was originally developed to screen protein expression libraries with 32P-labeled GST-fusion proteins16,17. The technique has now been used to study proteinprotein interactions, for example, receptorligand interactions, and to screen interacting partners in a library18. The major advantages of Far WB are that (i) it allows a prey protein to be endogenously expressed in cells; this is particularly meaningful when prey proteins are difcult to purify, and (ii) it enables one to determine whether two proteins directly

bind to each other and, if not, whether a third protein mediates the physical complexing between them. The major disadvantages are that at least one protein should be puried to certain amounts and that there may be some nonspecic binding. Far WB is widely used to (i) identify binding partners of a bait protein19,20; (ii) conrm one-to-one proteinprotein interactions resulting from highthroughput screening or other methods21,22. It can also be used to study the effect of post-translational modications on protein protein interactions, examine interacting domain using synthetic peptides as probes and identify proteinprotein interactions without using antigen-specic antibodies. Far WB can be performed in a laboratory in which facilities for protein purication and standard WB are available. Figure 1 shows a owchart of a Far WB protocol. Instead of detecting a blotted protein in WB based on its molecular weight, Far WB primarily detects the bait protein(s) that is immobilized in the positions where its binding partners (prey) are distributed according to their molecular weights. During the preparation of SDS-PAGE, proteins are typically reduced and denatured by treatment with the Laemmli sample buffer. Because many protein protein interactions depend on protein secondary and tertiary structures, which are disrupted under reducing and denaturing conditions, it is likely that few, if any, protein complexes could persist following the treatment with an SDS-PAGE sample buffer. In Far WB, proteins are usually denatured with guanidine or urea, and then renatured with gradient-reducing guanidine or urea, which allows proteins to recover their secondary and tertiary structures. To illustrate the Far WB protocol, we here utilize our studied proteins in our laboratory, polycystin-2 (PC2) and brocystin (FPC), which are mutated in autosomal dominant polycystic kidney disease (PKD) and autosomal recessive PKD, respectively. They do not bind directly to each other but in the presence of a third, intermediate protein, KIF3B, a motor subunit of kinesin-2, they form a complex22.

3278 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS

PROTOCOL
Experimental design Sample preparation. Prey protein: This can be puried protein or cell lysate of bacteria, yeast, insect or mammalian cells, which are endogenously/heterologously expressing the prey protein. Preparation of a recombination protein is not described here and can be found in literature23. There are no particular requirements with respect to how to prepare cell lysates19,24. Each Far WB experiment needs 0.21.0 mg of each puried protein or 10100 mg of cell lysates. The required amounts vary depending on protein purity and the expression level of the prey protein; over-expressed prey protein is preferred if the endogenous protein level is too low or if specic antibodies are not available. Bait protein can be puried from any organism. The protein partner that is the easiest to purify should be chosen as bait. Each Far WB experiment needs 110 mg of the bait protein, though the preybait binding strength and antibody sensitivity may affect the amount of the bait protein used. Standard controls. For each study, positive (a known interacting protein) and negative (known noninteracting proteins) controls should always be performed in parallel. Negative controls include, but are not limited to, the use of preparations lacking cell lysates, bait protein(s) or both. Usually, BSA is used in a negative control to replace the prey protein. Membrane choice. Nitrocellulose and polyvinylidene uoride (PVDF) are two commonly used membranes. Nitrocellulose membranes are cheaper than PVDF and are good for general purposes, but are far more fragile and do not stand up well for repeated probing. Because binding to membrane occurs primarily through hydrophobic bonds and PVDF is more hydrophobic, it would bind proteins more tightly than nitrocellulose membranes. Thus, PVDF is a better choice when the transferred protein is small and at low amounts. Denaturing/renaturing of prey proteins. After the separation on SDS-PAGE, the resulting proteins are denatured by SDS. Transferred proteins generally renature after SDS is eliminated during the transfer process. For Far WB, it is essential that at least the interaction domain of the prey protein is not disrupted by the transfer or is able to refold on the membrane to form a 3D structure comprising an intact interaction site. In the event that the protein is unable to refold to create an intact binding site, it is necessary to add a denaturation/renaturation step to the procedure, which is typically accomplished using guanidine hydrochloride. Alternatively, there are two approaches that do not include this denaturing/renaturing step: (i) In-Gel Far WB, in which the proteins are subjected to electrophoresis under native conditions, that is, nondenaturing and without reducing agent. The gel containing the prey protein is then incubated, usually with puried bait protein(s). Two major disadvantages of the approach are that the gel is too fragile to handle and that the bait protein(s) often has to
Prepare purified prey protein or cell lysate

Run SDS-PAGE

Transfer to membrane

Denature/renature

2007 Nature Publishing Group http://www.nature.com/natureprotocols

Blocking

Prepare purified bait protein(s)

Incubate membrane with the bait protein(s)

Detect the bait and prey proteins on the membrane by WB respectively

Figure 1 | A owchart for Far western blotting (WB).

be radiolabeled to increase sensitivity and reduce the required protein amount. (ii) Membrane-based Far WB without denaturing/renaturing, in which the proteins are subjected to electrophoresis under nondenaturing or denaturing conditions, and then transferred to the membrane for incubation with the puried bait protein(s). This method saves time and cost, but the prey protein may not correctly refold on the membrane. Detection systems. Chemiluminescence detection is one of the most sensitive and commonly used methods for WB. This method depends on incubation of the WB membrane with a substrate that produces luminescence when exposed to horseradish peroxidase (HRP) reporter conjugated on the secondary antibody. The light is then detected by photographic lm, and more recently by special charge-coupled device cameras that capture digital images of the WB. The image is analyzed by densitometry, which evaluates the relative amount of protein staining and quanties the results in terms of optical density. Blots detected with the chemiluminescence method are easily stripped for subsequent reprobing with additional antibodies, such as those against bait and/or prey. Other detection methods include (i) colorimetric detection, such as BCIP/NBT-blue liquid substrate system (AP conjugation) or TMB (HRP conjugation), (ii) radioactive detection and (iii) uorescence detection.

MATERIALS

. Puried (bait) proteins (see EXPERIMENTAL DESIGN for further details of . Prey protein (in crude extracts or puried, see EXPERIMENTAL DESIGN
for further details of sample preparation) sample preparation)

REAGENTS

. Acrylamide:bis-acrylamide (29:1) (Bio-Rad, cat. no. 161-0156) . Guanidine hydrochloride (Sigma, cat. no. G4505-500G) . N,N,N,N-Tetramethylethylenediamine (TEMED; Sigma, cat. no. T928125ML) ! CAUTION Harmful if inhaled or swallowed; can cause skin and eye irritation.

NATURE PROTOCOLS | VOL.2 NO.12 | 2007 | 3279

PROTOCOL
TABLE 1 | AC buffer with varying concentrations of guanidineHCl used in denaturing and renaturing. Concentration of guanidineHCl (M) Glycerol (ml) 5 M NaCl (ml) 1 M Tris, pH 7.5 (ml) 0.5 M EDTA (ml) 10% Tween-20 (ml) GuanidineHCl (8 M) (ml) Milk powder (g) 1 M DTT (ll) ddH2O (ml) Total volume (ml) 6 2.5 0.5 0.5 0.05 0.25 18.75 0.5 25 2.45 25 30 min/room temperature (RT) 3 2.5 0.5 0.5 0.05 0.25 9.30 0.5 25 12.82 25 30 min/RT 1 2.5 0.5 0.5 0.05 0.25 3.13 0.5 25 18.07 25 30 min/RT 0.1 2.5 0.5 0.5 0.05 0.25 0.31 0.5 25 20.89 25 30 min/4 1C 0 2.5 0.5 0.5 0.05 0.25 0 0.5 25 21.20 25 1 h overnight/4 1C

2007 Nature Publishing Group http://www.nature.com/natureprotocols

Time/temperature

. Ammonium persulfate (APS), 10% solution (Sigma, cat. no. A3678-100G)

! CAUTION Harmful if swallowed. m CRITICAL Prepare fresh as it decomposes on exposure to water. . Nitrocellulose or PVDF membrane (Bio-Rad, cat. no. 162-0094; see EXPERIMENTAL DESIGN for further details of membrane choice) . Primary (against bait or prey protein) and secondary (HRP conjugate) antibodies (primary antibodies monoclonal anti-GST and anti-PC2: custom made; rabbit polyclonal anti-GFP: Clontech, cat. no. 632460; secondary antibodies goat anti-mouse and goat anti-rabbit: Chemicon, cat. no. AQ502P and cat. no. AQ132P, respectively.) . ECL detection kit (Amersham Biosciences, cat. no. RPN-2106) . Pre-stained protein ladder (Bio-Rad, cat. no.161-0374) . Tris base (Invitrogen, cat. no. 15504-020-1KG) . Glycine (Bio-Rad, cat. no. 161-0724-2KG) . SDS (Bio-Rad, cat. no. 161-0302-1KG) ! CAUTION Harmful if inhaled or swallowed; can cause skin and eye irritation. . Skim milk powder (local supermarket) . Tween-20 (Sigma, cat. no. P7949-100ML) . DTT (Bio-Rad, cat. no. 161-0611-5G) . Glycerol (Invitrogen, cat. no. 15514-011) . b-Mercaptoethanol (Sigma, cat. no. M7154-100ML) ! CAUTION Highly toxic. Wear suitable protective clothing, gloves and eye/face protection. It is harmful if inhaled or swallowed, and toxic when in contact with skin. . Bromophenol blue (Sigma, cat. no. B0126-25G) . Methanol (Fisher Scientic, cat. no. A935-4) ! CAUTION Flammable. . KH2PO4 (Sigma, cat. no. P9791-1KG) . Na2HPO4 (Sigma, cat. no. S3264-1KG) . Ponceau S (Sigma, cat. no. 81462-1L) . Separating gel (see REAGENT SETUP) . Stacking gel (see REAGENT SETUP) . Protein loading dye (see REAGENT SETUP) . TrisGlycine running buffer (see REAGENT SETUP) . Transfer buffer (see REAGENT SETUP) . Denaturing and renaturing buffers (see REAGENT SETUP) . Protein-binding buffer (see REAGENT SETUP) . PBST buffer (see REAGENT SETUP) . Re-blot Plus Strong Solution (Chemicon, cat. no. 2504-50ML) EQUIPMENT . Vertical electrophoresis apparatus (Mini-PROTEAN 3; Bio-Rad, cat. no. 165-3301) . Power pack (Powerpac 3000; Bio-Rad, cat. no. 164-5070) or equivalent

. Transfer system (Mini Trans-Blot Cell; Bio-Rad, cat. no. 170-3930) . Petri dish (100 15 13 mm3; BD Biosciences, cat. no. 351112) . Whatman chromatography paper (Fisher Scientic, cat. no. 05-717-3A) . X-ray lm (Kodak, cat. no. 8377616) or equivalent, developer or phosphor
imaging plate and detection system (Fuji BAS-MS; Fuji) or equivalent REAGENT SETUP Separating gel 8% Separating SDS-PAGE gel mixture (29:1) acrylamide: bis-acrylamide, 375 mM TrisCl (pH 8.8), 0.1% SDS, 0.1% APS and 0.06% TEMED. The gel mixture should be freshly prepared. m CRITICAL The choice of the percentage of the separating gel depends on the molecular weight(s) of prey protein(s); generally, the higher the molecular mass, the lower the percentage of the separating gel used. ! CAUTION Acrylamide and bis-acrylamide are highly toxic and are potential human carcinogens and teratogens. Purchase as stock solutions to avoid exposure to powder, which could easily be dispersed during weighing, for example. ! CAUTION SDS is a respiratory, skin and eye irritant. It is harmful if swallowed, inhaled or absorbed through the skin. Stacking gel 4% Acrylamide:bis-acrylamide (29:1), 125 mM TrisCl (pH 6.8), 0.1% SDS, 0.1% APS and 0.1% TEMED. The gel mixture should be freshly prepared. ! CAUTION Acrylamide and bis-acrylamide are highly toxic and are potential human carcinogens and teratogens. Purchase as stock solutions to avoid exposure to powder, which could easily be dispersed during weighing, for example. ! CAUTION SDS is a respiratory, skin and eye irritant. It is harmful if swallowed, inhaled or absorbed through the skin. Protein loading dye 50 mM TrisHCl (pH 6.8), 2% SDS, 10% glycerol, 1% b-mercaptoethanol, 12.5 mM EDTA and 0.02% bromophenol blue. This loading dye can be stored at room temperature (RT, 2023 1C) for up to 6 months. TrisGlycine running buffer 25 mM TrisCl (pH 8.3), 190 mM Glycine, 1% SDS. This buffer can be stored at RT for up to 3 months. Transfer buffer 10% Methanol, 24 mM Tris, 194 mM Glycine. The buffer should be freshly prepared and kept refrigerated before use. Denaturing and renaturing buffers: AC buffer 100 mM NaCl, 20 mM Tris (pH 7.6), 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, 2% skim milk powder and 1 mM DTT (see Table 1). These solutions should be freshly prepared. Protein-binding buffer 100 mM NaCl, 20 mM Tris (pH 7.6), 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, 2% skim milk powder and 1 mM DTT. The solution should be freshly prepared. PBST buffer 4 mM KH2PO4, 16 mM Na2HPO4, 115 mM NaCl (pH 7.4) and 0.05% Tween-20. Usually prepare 10 PBST, and store at RT for up to 3 months, and dilute to 1 before use.

PROCEDURE Sample preparation 1| Samples from any source can be used in this experiment (See EXPERIMENTAL DESIGN for further details of sample preparation). We used lysates of cultured mammalian cells over-expressing a prey protein (the C terminus of FPC with a GFP tag (GFPFPCC)), and bacterially puried bait proteins (the C termini of KIF3A (KIF3AC) and KIF3B (KIF3BC) proteins with His tags, and those of PC2 and FPC with GST tags; see Fig. 2). m CRITICAL STEP The most critical step is to have sufcient amounts of prey proteins (expressed either in crude lysates (10100 mg) or in puried forms (0.21 mg)) and of the bait proteins in puried forms (110 mg). PAUSE POINT Cell lysates and puried proteins can be stored at 80 1C for several months.
3280 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS

PROTOCOL
SDS-PAGE separation of prey proteins 2| Pour the separating gel (see REAGENT SETUP), using 10 ml per gel, leave enough room for a stacking gel, overlay with water and let it polymerize for 30 min. 3| Remove the aqueous layer from the separating gel and pour the stacking gel (see REAGENT SETUP), B3 ml per gel, on top of the separating gel. 4| Insert a comb, let it polymerize for at least 30 min. 5| Clamp the gel onto the electrophoresis tank. m CRITICAL STEP Commercially ready-to-use gel can be alternatively used. 6| Adjust prey protein concentration so that a suitable amount of protein is loaded onto the gel. Usually, 0.21 mg of puried protein is required, or 10100 mg of cell lysates. m CRITICAL STEP Concentrating the samples by SpeedVac may be necessary if the volume is too large for loading.

a
C C KI F3 B KI F3 A is is H

b
G ST G PC S T 2C -F PC C G ST H

c
G FP G FP BS A -F PC C

kDa 75 50 50 37 37 25 CBB

100 75 50 37 25 WB: GST WB: GFP

2007 Nature Publishing Group http://www.nature.com/natureprotocols

Figure 2 | Expression and purication of polypeptides. The soluble C termini of KIF3A (KIF3AC, aa 403702), of KIF3B (KIF3BC, aa 407747), of polycystin-2 (PC2C, aa 682968) and of brocystin (FPCC, aa 38824074) were used in this study. (a) His-tagged KIF3BC and KIF3AC proteins were puried from bacteria and stained by Coomassie brilliant blue (CBB). (b) Escherichia coli extracts expressing glutathione S-transferase (GST) alone, GST-PC2C or GST-FPCC, were subjected to western blotting (WB) with an antibody against GST. (c) GFP and GFPFPCC were stably expressed in MDCK cells. Shown are WB data using an anti-GFP antibody. BSA acts as a negative control.

7| Apply the loading buffer (nal 1, see REAGENT SETUP) to the prey protein samples (maximum volume r 50 ml), boil the samples for 5 min, and set on ice right after. In our experiments, we used 0.5 mg BSA and 50 mg cell lysates over-expressing GFP as negative controls or GFPFPCC as an experimental prey protein (Fig. 2c). Since KIF3AC and KIF3BC associate with each other through their coiled-coil structure, we have used them as a positive control where we used puried KIF 3AC (0.5 mg) as prey and separated on gel, and puried KIF3BC (5 mg) as bait. m CRITICAL STEP Optionally, duplicate or triplicate gels/membranes can be prepared to allow the use of one each for Far WB to detect bait protein (Step 22) and standard WB with an antibody against the prey protein (Step 23), which helps in determining whether bait and prey are on the same spot (rather than using the same membrane for both these procedures). A third membrane may also be useful for staining with Ponceau S (Step 12). 8| Load samples onto the gel (sample volume should not be 450 ml for a 1.5 mm 10-well gel) along with positive and negative controls, and protein ladder. 9| Run gel at 80 V for 20 min, then increase to 125 V for a period of time depending on the position of the prey proteins on the gel, which is monitored by a prestained protein ladder. The ideal pattern is that prey proteins stay in the middle of the gel. Transfer of proteins onto the membrane 10| Prechill the transfer buffer and submerge the membrane in the transfer buffer before use. ? TROUBLESHOOTING 11| Use the traditional sandwich method24 to transfer proteins from gel to membrane at 100 V for 2 h at 4 1C. ? TROUBLESHOOTING 12| Stain the membrane with Ponceau S24 to check whether proteins have been transferred from the gel to the membrane. This step will not affect the following assay. m CRITICAL STEP Alternatively, standard WB24 with an anti-prey antibody can be performed to check whether proteins are equally transferred to the membrane. Use equal amounts of BSA (negative control) and cell lysates to run SDS-PAGE, transfer to the nitrocellulose membrane and then detect with WB (Fig. 2c). This must be done on duplicate membranes for standard WB and Far WB. It is best to carry out standard WB before the rest of the Far WB procedure to ensure the proteins have been transferred to the membrane. Alternatively, perform in Step 23, as described. ? TROUBLESHOOTING Denaturing/renaturing of proteins on the membrane 13| Denature and renature proteins on the membrane in AC buffer by gradually reducing the guanidineHCl concentration (see Table 1). Briey, denature proteins by incubating the membrane in the AC buffer containing 6 M guanidineHCl for 30 min at RT. Then wash with the AC buffer containing 3 M guanidineHCl for 30 min at RT. This is followed by washing with the AC buffer containing 0.1 M and no guanidineHCl AC buffer at 4 1C, for 30 min and 1 h, respectively.

NATURE PROTOCOLS | VOL.2 NO.12 | 2007 | 3281

PROTOCOL
m CRITICAL STEP Completely renaturing proteins on the membrane is critical for subsequent proteinprotein binding assays. In order to keep proteins in proper renaturing states, incubation of the membrane with 0.1 M and no guanidineHCl AC buffers at 4 1C is strongly recommended. Also, the time of incubation with the guanidineHCl-free AC buffer should be at least 1 h. PAUSE POINT The last renaturing step with the guanidineHCl-free AC buffer can last overnight at 4 1C. ? TROUBLESHOOTING Blocking 14| Block the membrane with 5% milk in the PBST buffer for 1 h at RT. m CRITICAL STEP After transferring and renaturing proteins on the membrane, use 5% milk to block nonspecic proteins on the membrane, in contrast to 2% used in the previous steps. Determination of an effective blocking buffer should rstly be made empirically. Often, skim milk and BSA are used as a starting point. Insufcient blocking may result in high background, whereas prolonged blocking could result in a weak or masked signal. Protein renaturing also appears to occur during the blocking step, so it is important to optimize the blocking conditions to obtain the best signal:noise ratio for each application and not to deviate from the method. Incubation of the membrane with puried interacting (bait) protein(s) 15| Incubate the membrane with puried bait protein(s) (e.g., total 5 mg, 1 mg ml1) in the protein-binding buffer (or an equivalent buffer) overnight at 4 1C. In our experiments, we applied 5 mg puried GST-PC2C, His-KIF3AC or His-KIF3BC in a 5 ml reaction buffer. m CRITICAL STEP The strength of proteinprotein interaction may vary as a function of pH, salt concentration and certain co-factors during the incubation of bait protein(s). It is important that conditions do not change throughout the procedure to maintain the interaction until it is detected. This may affect the formulation of a washing buffer used. To save the proteins used, minimize the binding buffer volume. PAUSE POINT Incubation with puried interacting proteins can be carried out overnight at 4 1C if time is allowed. We have compared different incubation conditions (1 h at 37 1C, 3 h at RT and overnight at 4 1C) and found no signicant difference. However, incubation overnight at 4 1C is recommended, as the result is more reproducible under this condition between probing steps. Detection of bait proteins bound to prey proteins on the blot 16| Wash off unbound bait protein(s) with PBST buffer three times, each for 10 min. 17| Incubate the blot with an appropriate diluted primary antibody for 1 h at RT in 3% milk in the PBST buffer. In our experiments, we used an antibody against PC2 (1A11) at 1:2,000 dilution. PAUSE POINT Incubation with the primary antibody can be carried out overnight at 4 1C, which has been found to be reliable and reproducible in our laboratory. 18| Wash with the PBST buffer three times, each for 10 min. 19| Incubate with a secondary antibody for 1 h at RT. In our experiments, we used an HRP-conjugated anti-mouse IgG antibody at 1:5,000 dilution. 20| Wash with the PBST buffer three times, each for 10 min, and then rinse with PBS buffer for 5 min. 21| Perform chemiluminescent detection of the bound bait protein using an ECL kit, according to the manufacturers instruction. m CRITICAL STEP See EXPERIMENTAL DESIGN for details of alternative detection systems. 22| Develop X-ray lm immediately to visualize the position of the bait protein. ? TROUBLESHOOTING 23| Strip the probe with the Re-Blot Plus Strong solution for 20 min at RT, and use the same membrane to perform a standard WB24, using an antibody against the prey protein, to determine whether bait and prey are at the same position on the membrane.
3282 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS

2007 Nature Publishing Group http://www.nature.com/natureprotocols

Bait proteins added

BS A G FP G FP -F

BS A G FP G FP -F

FP

BS

kDa 100 100 PC2C + KIF3AC 75 50 37 25

100 PC2C + KIF3BC 75 50 37 25 1 2 3

PC2C

75 50 37 25

Figure 3 | Far western blotting (WB):KIF3BC bridges the association between PC2C and FPCC. Lysates of MDCK cells expressing GFPFPCC or GFP and BSA were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Proteins were denatured, renatured and then incubated with puried GST-PC2C together with (a) none, (b) KIF3AC or (c) KIF3BC. After washing, bound proteins were detected with a PC2 antibody. BSA and GFP vector (lanes 1 and 2) serve as negative controls. The arrow points to an extra band (lane 3, c) not seen in the panels of (a) and (b), corresponding to the binding of PC2C to the site of FPCC (shown in Fig. 2c). GST, glutathione S-transferase.

FP

-F PC

PC

PC

PROTOCOL
Results analysis 24| Compare the results of Far WB (Step 22) and standard WB (Step 23), and judge whether the bait and prey proteins are located on the same spots. For example, we compared Figures 2c and 3, which show the positions of the prey and/or bait proteins, and concluded that FPCC associates with PC2C in the presence of KIF3BC. TIMING Steps 18, sample preparation and loading: 30 min to 1 h Step 9, electrophoresis: 3 h Steps 1012, electroblotting: 2 h Steps 13, denaturing/renaturing: 3 hovernight Step 14, blocking: 1 h Step 15, protein binding: 3 h-overnight Steps 1623, detection of proteins: 4 hovernight Step 24, results analysis: 30 min ? TROUBLESHOOTING Troubleshooting advice can be found in Table 2.
TABLE 2 | Troubleshooting table. Step 1012 Problem Incomplete transfer of proteins to membrane Possible reasons Polyvinylidene uoride (PVDF) was not made wet Inappropriate transfer buffer used Transfer time is not enough Solutions Presoak PVDF membrane with methanol for 1 min Fresh buffer should be used for transfer process Add SDS (nal 0.1%) into transfer buffer Increase transfer time Use low temperature and low voltage for transfer Wash membrane with guanidiniumHCl-free solution between each step Renature at 4 1C for overnight Check protein concentration and quality of both bait and prey by SDS-PAGE staining and standard western blotting (WB) Increase the prey and bait protein amounts Check primary and secondary antibodies by WB Check the antibody by standard WB, or use an alternative antibody Reduce the concentration and incubation time of antibodies Reduce the prey and bait protein amounts Keep membrane wet all the time

2007 Nature Publishing Group http://www.nature.com/natureprotocols

13

Proteins are not completely renatured

A trace of guanidinium hydrochloride was left in the nal renature solution Renature time is not enough Not enough protein used

22

No signal

Proteins do not refold properly Antibody does not work well Too many bands on the blot (high background) signal Other binding partners of the bait are present Blocking is not enough Antibody is not correctly used Membrane dried up during process

ANTICIPATED RESULTS A successful Far WB appears to be similar to a standard WB. However, the bait protein(s), as well as a prey protein, is detected on the spots where the prey proteins are located (according to the molecular weight of the prey but not of the bait). In addition, there are often some nonspecic bands on the blot, which probably correspond to some other binding partners of the bait protein(s) that are present in the cell lysates. As shown in Figure 3, we found that our studied protein PC2 associates with brocystin through the intermediate protein KIF3B, but not directly or through KIF3A. Far WB procedures must be performed with care to preserve as much as possible the native conformation of the proteins under study. Denatured proteins may not be able to interact, resulting in a failure to identify an interaction. On the other hand, proteins in non-native conformations may interact with nonspecic proteins, resulting in false-positive interactions. Denaturing may have more impact on prey proteins, because they undergo preparative processing steps for Far WB. This is not to imply

NATURE PROTOCOLS | VOL.2 NO.12 | 2007 | 3283

PROTOCOL
that identication of valid interactions is not possible but only to stress the importance of an appropriate validation and the use of controls. As discussed earlier, experimental conditions should be optimized individually.

ACKNOWLEDGMENTS This work was supported by the Canadian Institutes of Health Research and the Kidney Foundation of Canada. X.-Z.C. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research. Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Fu, H. Protein-Protein Interactions: Method and Application. (Humana Press, Totowa, New Jersey, 2004). 2. Golemis, E.A. & Adams, P.D. ProteinProtein Interactions: A Molecular Cloning Manual 2nd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2005). 3. Protein interaction technologies. In Molecular Cloning: A Laboratory Manual 3rd edn. Ed: Sambrook, J. & Russell, D.W. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). 4. Dmitrova, M. et al. A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol. Gen. Genet. 257, 205212 (1998). 5. Vasavada, H.A., Ganguly, S., Germino, F.J., Wang, Z.X. & Weissman, S.M. A contingent replication assay for the detection of protein-protein interactions in animal cells. Proc. Natl. Acad. Sci. USA 88, 1068610690 (1991). 6. Fields, S. & Song, O. A novel genetic system to detect protein-protein interactions. Nature 340, 245246 (1989). 7. Gordon, G.W., Berry, G., Liang, X.H., Levine, B. & Herman, B. Quantitative uorescence resonance energy transfer measurements using uorescence microscopy. Biophys. J. 74, 27022713 (1998). 8. Peger, K.D., Seeber, R.M. & Eidne, K.A. Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat. Protoc. 1, 337345 (2006). 9. Ye, P. et al. Gene function prediction from congruent synthetic lethal interactions in yeast. Mol. Syst. Biol. 1, 2005 (2005). 10. Rigaut, G. et al. A generic protein purication method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 10301032 (1999). 11. Smith, E.A. & Corn, R.M. Surface plasmon resonance imaging as a tool to monitor biomolecular interactions in an array based format. Appl. Spectrosc. 57, 320A332A (2003).

2007 Nature Publishing Group http://www.nature.com/natureprotocols

12. Jones, R.B., Gordus, A., Krall, J.A. & MacBeath, G. A quantitative protein interaction network for the ErbB receptors using protein microarrays. Nature 439, 168174 (2006). 13. Hall, R.A. Studying protein-protein interactions via blot overlay or far western blot. In ProteinProtein Interactions: Methods and Applications. p. 167174 (ed. Fu, H.) (Humana Press, Totowa, New Jersey, 2004). 14. Vikis, H.G. & Guan, K.L. Glutathione-S-transferase-fusion based assays for studying protein-protein interactions. In ProteinProtein Interactions: Methods and Applications. p. 175186 (ed. Fu, H.) (Humana Press, Totowa, New Jersey, 2004). 15. Wang, B., Pelletier, J., Massaad, M.J., Herscovics, A. & Shore, G.C. The yeast splitubiquitin membrane protein two-hybrid screen identies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphataselike B. Mol. Cell Biol. 24, 27672778 (2004). 16. Kaelin, W.G. Jr. et al. Expression cloning of a cDNA encoding a retinoblastomabinding protein with E2F-like properties. Cell 70, 351364 (1992). 17. Blackwood, E.M. & Eisenman, R.N. Max: a helix-loop-helix zipper protein that forms a sequence-specic DNA-binding complex with Myc. Science 251, 12111217 (1991). 18. Kaido, M. et al. Downregulation of the NbNACa1 gene encoding a movementprotein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana. Mol. Plant Microbe Interact. 20, 671681 (2007). 19. Cantor, S.B. et al. BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Cell 105, 149160 (2001). 20. Gupta, R. et al. FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the singlestranded DNA binding protein. Blood 110, 23902398 (2007). 21. Feldman, M., Zusman, T., Hagag, S. & Segal, G. Coevolution between nonhomologous but functionally similar proteins and their conserved partners in the Legionella pathogenesis system. Proc. Natl. Acad. Sci. USA 102, 1220612211 (2005). 22. Wu, Y. et al. Kinesin-2 mediates physical and functional interactions between polycystin-2 and brocystin. Hum. Mol. Genet. 15, 32803292 (2006). 23. Cutler, P. Protein Purication Protocols. (Humana Press, Totowa, New Jersey, 2003). 24. Sambrook, J., Fritsch, E.F. & Maniatis, T. Detection and analysis of proteins expressed from cloned genes. In Molecular Cloning: A laboratory Manual 2nd edn. 18.1918.74 (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

3284 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS