Detection of Protein-Protein Interactions UNIT 20.

5
by Coprecipitation
Coprecipitation of proteins from whole-cell extracts is a valuable approach to test for
physical interactions between proteins of interest. When a precipitating antibody is used,
this method is referred to as co-immunoprecipitation. Coprecipitation can be used to
study interactions between known proteins under a variety of conditions and as a means
of identifying components of a complex. Coprecipitation may be the single method of
choice, or may be used in combination with other methods that detect protein-protein
interactions, such as two-hybrid analysis and copurification schemes (UNIT 20.1), and tests
of physical associations using purified proteins.

This unit describes basic approaches to immunoprecipitating tagged proteins from whole-
cell extracts. The approaches described can be adapted for other systems. In a typical
experiment, as described here, cells are lysed and a whole-cell extract is prepared under
nondenaturing conditions (see Strategic Planning). The protein is precipitated from the
lysate with a solid-phase affinity matrix, and the precipitate is tested for the presence
of a second specifically associated protein (see Basic Protocol and Alternate Protocol).
The approach can be used for native or epitope-tagged proteins for which antibodies are
available, or for recombinant proteins that have been engineered to bind with high affinity
to a molecule that can be coupled to a solid-phase matrix (see Strategic Planning). The
presence of an associated protein is detected by separating the precipitated proteins by
SDS-PAGE (UNIT 10.2A) and then immunoblotting (UNIT 10.8) with a second antibody that
recognizes the putative associated protein. Controls to test specificity of interaction are
crucial (see Strategic Planning).

For additional background reading, the user should consult UNIT 10.16 for a theoretical
discussion of immunoprecipitation; see Chapter 11 for principles of antibody production
and immunoassays; see UNITS 10.15, 20.2 & 20.3 for approaches to tagging proteins; and see
Chapter 13 for transformation and propagation of S. cerevisiae. For an in-depth review of
immunoprecipitation techniques, see Harlow and Lane (1988). For an in-depth review of
coprecipitation and other approaches to detect protein-protein interactions, see Phizicky
and Fields (1995), Toby and Golemis (2001), Auerbach et al. (2002), and Gavin and
Superti-Ferga (2003).

STRATEGIC PLANNING
Detecting the Proteins in Question
The first step is to generate reagents that detect the two proteins in the coprecipitate
under nondenaturing conditions. If antibodies are available that can immunoprecipitate
the proteins under nondenaturing conditions, then they can be used. Alternatively, the
proteins can be differentially tagged in a variety of ways to allow their detection with
commercially available antibodies or other affinity reagents. The tagged proteins are then
introduced into the host organism using expression vectors. All tagged proteins must be
assessed for function in vivo.

A frequently used option is to add a short peptide or epitope that is recognized by a

commercially available high-affinity monoclonal antibody (mAb; UNIT 10.15). The epitope
is typically added at the amino or carboxyl terminus, although internal positions that
do not disrupt function can also be used. Two frequently used epitopes are derived
from influenza hemagglutinin protein (HA) and human c-Myc and are recognized by Analysis of
Protein
Interactions
Contributed by Elaine A. Elion 20.5.1
Current Protocols in Molecular Biology (2006) 20.5.1-20.5.10
Copyright

C 2006 by John Wiley & Sons, Inc.
Supplement 76
 high-affinity mAbs (12CA5 and 9E10, respectively; Kolodziej and Young, 1991). Others
such as FLAG, the leader peptide of the gene 10 product of bacteriophage T7 (Knappik
and Pluckthun, 1994; Witzgall et al., 1994), are also available. The choice of epitope
may be dictated by its amino acid composition. It is often useful to insert tandem copies
of the epitope in order to increase sensitivity. The number of additional tandem copies
can range widely from one (Field et al., 1988) to several (e.g., three; Tyers et al., 1993)
to many (e.g., nine; Feng et al., 1998).

Proteins can also be fused to small proteins or peptides that have high affinity to small
molecules that can be attached to a solid support. This is a particularly valuable ap-
proach when the protein to be precipitated comigrates with immunoglobulin heavy or
light chains in an SDS-polyacrylamide gel. Such alternative tagging methods include
fusion to glutathione-S-transferase (to allow purification by a glutathione affinity ma-
trix; UNIT 16.7), maltose-binding protein (to allow purification by a maltose affinity ma-
trix; UNIT 16.6), polyhistidine (to allow purification by nickel affinity matrix; Bornhorst
and Falke, 2000), or strep-tag (to allow purification by streptavidin matrix; Skerra and
Schmidt, 2005). Tandem affinity protein (TAP) tags made of two IgG-binding domains of
Staphylococcus aureus protein A and a calmodulin-binding peptide separated by a TEV
protease cleavage site (Puig et al., 2001) have also been successfully used to copurify
proteins, especially in yeast (Gavin and Superti-Furga, 2003). Other TAP tags have been
described, including a TAP tag of three tandem FLAG epitopes and six histidines that
may be more efficient in some organisms (Yang et al., 2006). A reference for identifying
sources of commercially available antibodies and vectors encoding various tags can be
found in the BioSupplyNet Source Book (2005). See Chapter 11 and Harlow and Lane
(1998) for the generation and purification of specific antibodies. The American Type Cul-
ture Collection (ATCC) and European Collection of Animal Cell Cultures can be sources
for hybridoma cell lines. Numerous genomic coprecipitation and two-hybrid studies have
been done in a variety of organisms, so it is wise to check available databases for the
current repertoire of interactions for a particular protein (e.g., Gavin and Superti-Furga,
2003, lists a number of such studies). Useful summaries of protein-protein interactions
along with references can be found at Proteome Bioknowledge Library (Biobase Biolog-
ical Databases, http://www.biobase.de; formerly Proteome, Inc., a subsidiary of Incyte),
which is available by institutional or individual subscription.

Preparing Whole-Cell Extracts

The second step in a successful coprecipitation is generating whole-cell extracts that
optimize the yield and activity of the proteins to be analyzed, using lysis buffer conditions
that permit recognition of the proteins by the affinity matrix. The yield of total protein
in a whole-cell extract is not always a reliable indicator of the relative yield and activity
of specific proteins, so it is wise to verify both parameters at the onset of an experiment
before proceeding with the coprecipitation. Yield and activity can be affected by a number
of factors (see Chapter 10; see Harlow and Lane, 1988). Small variations in the relative
amounts of salt and detergents in the lysis buffer can have large effects on yield and
activity, as can the speed and efficiency of cell breakage. Both factors are particularly
important for less soluble proteins that associate with macromolecular structures such
as membranes or cytoskeleton. In addition, global inhibition of proteolysis through the
inclusion of multiple classes of protease inhibitors may be essential.

Methods for preparing whole-cell extracts from yeast (UNIT 13.13), E. coli (UNITS 16.1-16.8),
insect cells (UNIT 16.11), and mammalian cells (UNITS 16.12-16.18) can be found elsewhere
Detection of in this manual, and specifics will not be discussed here. In general, the lysis buffer
Protein-Protein
Interactions by conditions are not very different from the coprecipitation conditions. It is recommended
Coprecipitation that the investigator begin by comparing small-scale extract preparations that vary the
20.5.2
Supplement 76 Current Protocols in Molecular Biology
amount of salt and nonionic detergent. As a starting point, a basic lysis buffer might
contain the following components.

Basic components. Basic components include a buffering agent (such as 50 mM TrisCl,

pH 7.5), a small amount of nonionic detergent (such as 0.1% [v/v] Triton X-100), salt
(such as 100 mM NaCl), a reducing agent (such as 1 mM DTT), and 10% (v/v) glycerol
as stabilizer.

Protease inhibitors. Protease inhibitor cocktails are described in UNIT 13.13 and are also
commercially available. A reasonable starting point would be to include 5 g/ml each
chymostatin, pepstatin A, leupeptin, and antipain, as well as 1 mM phenylmethysulfonyl
fluoride and 1 mM benzamidine.

Chelating agents. EGTA (15 mM) is commonly included to chelate divalent metal
ions that are essential for metalloprotease activity. Because EGTA also inhibits other
metal-dependent enzymes, it may be omitted, combined with the addition of a needed
metal ion, and/or substituted with EDTA.

Phosphatase inhibitors. If the phosphorylation state of the proteins in question is impor-

tant, a mixture of phosphatase inhibitors should also be included in the lysis buffer. A
starting mixture could contain 2.5 mM each meta- and ortho-vanadate, 10 mM NaF, and
10 mM -glycerol phosphate.

Simple modifications of this initial buffer include varying the amount of NaCl (from
0 to 500 mM) and of Triton X-100 (from 0% to 1%). The investigator may choose to
compare different means of breaking the cells (for example, glass-bead breakage versus
liquid nitrogen/grinding methods for yeast cells; UNIT 13.13).

Total protein concentration in the whole-cell extract is generally assayed by using the
Bio-Rad protein assay and calculating protein concentration (UNIT 10.1A). Extracts should
be tested for the amount of each specific protein by immunoblot analysis (UNIT 10.8),
analyzing 25 to 75 g of total protein. In general, it is best to test for the presence of a
second established protein (such as a housekeeping enzyme, cytoskeletal or ribosomal
protein, or a previously defined component in the pathway being studied) as an internal
control for normalization and as a positive control for the immunoblot. The amount of
specific protein in the whole-cell extract is compared to the amount that is recovered by
precipitation with an affinity matrix.

Control Tests for Specicity of Interaction

Controls are essential to verify that the antibodies and protein-protein interactions are
specific. Proper controls are simplest to set up when the proteins are differentially tagged.
In this instance, two parallel extracts are prepared from strains that contain each protein
lacking the tag in the presence of the second tagged protein. An example is shown in
the idealized gel in Figure 20.5.1, which includes lanes containing untagged protein 1 +
tagged protein 2 and tagged protein 1 + untagged protein 2. If the antibodies are specific,
untagged protein 1 will not immunoprecipitate. The presence of untagged protein 1 in
the immunoprecipitate will indicate that it binds the affinity matrix nonspecifically. If the
interaction between proteins 1 and 2 is specific, then tagged protein 2 will be present in
the immunoprecipitate of tagged protein 1, but not in its absence.

If antibodies to native proteins are used, it is necessary to compare extracts made from
strains harboring deletions of the proteins in question to test for the specificity of the
antibody and the interaction. However, this is obviously possible only if the deletions Analysis of
do not cause inviability. If deletion mutations cannot be used, a common approach is Protein
Interactions
to show that the preimmune serum or an antibody not known to be specific to either of
20.5.3
Current Protocols in Molecular Biology Supplement 76
 the proteins in question does not coprecipitate them in a parallel experiment. However,
the latter two controls do not rule out the possibility that the antibody is precipitating the
protein in question through an indirect association.

It is also essential to compare the amount of coprecipitated protein with the amounts
of the two proteins in question in the whole-cell extract. This allows one to determine
whether apparent differences in the ability of the two proteins to coprecipitate are a sec-
ondary consequence of the relative abundance of the proteins. This control is particularly
important when an interaction has been established and the investigator wishes to search
for regulatory changes in association apart from changes in abundance.

Detection of
Protein-Protein Figure 20.5.1 Flowchart for the coprecipitation of two proteins that have been differentially
Interactions by tagged and introduced into the host organism. Ig h and Ig l, immunoglobulin heavy and light
Coprecipitation chains; NT, no tag.

20.5.4
Supplement 76 Current Protocols in Molecular Biology
COPRECIPITATING PROTEINS WITH PROTEIN A/GSEPHAROSE BASIC
PROTOCOL
Once the conditions of extract preparation have been established (see Strategic Planning),
the next step is to test for coprecipitation of the specific proteins. This protocol describes a
standard coprecipitation procedure that uses an antibody coupled to protein ASepharose
or protein GSepharose. An alternative coprecipitation method that uses GST coupled
to glutathione-agarose is also provided (see Alternate Protocol). It is essential to keep all
buffers and tubes cold by using an ice bath and a refrigerated centrifuge. The conditions
of coprecipitation match the conditions of the lysis buffer described above.

Materials
Whole-cell extract (see Strategic Planning)
Antibody against protein or epitope of interest
5 M NaCl
Co-immunoprecipitation buffer (see recipe)
Protein A/GSepharose slurry (see recipe)
2 sample buffer for SDS-PAGE (UNIT 10.2A)
Test tube rotator
20-ml syringe and 18-G needle
Hamilton syringe
Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and immunoblotting
(UNIT 10.8)
1. Prepare duplicate samples in microcentrifuge tubes on ice:
0.5 to 1 mg whole-cell extract
1 g antibody
5 M NaCl to equalize at 100 mM NaCl
Co-immunoprecipitation buffer to 0.5 ml final volume.
Adjust buffer by adding a divalent cation if necessary for activity of the protein in question
(see Strategic Planning).

2. Invert tube gently several times and incubate on ice for 90 min with occasional tube
inversion.
It is recommended that the investigator begin with a 90-min incubation. However, this
incubation step can be shortened or lengthened.

3. Microcentrifuge 10 min at maximum speed, 4 C, to pellet nonspecific eggregates.

Transfer supernatant to a new microcentrifuge tube.
4. Add 50 l of protein A or protein GSepharose slurry (25 to 30 l bead volume).
Be sure to evenly suspend the slurry before distributing it to the samples.
Protein A has been used more frequently for historical reasons; however, protein G binds
a broader range of Ig subtypes at higher efficiency. See UNIT 11.11 for a description of
their relative binding capacities.

5. Rotate tube gently at 4 C for 30 to 60 min.

Rocking is much less efficient and should be avoided.

6. Gently pellet protein A/GSepharose by centrifuging 30 sec at 1000 rpm in a tabletop

centrifuge, 4 C.

Analysis of
Protein
Interactions

20.5.5
Current Protocols in Molecular Biology Supplement 76
 7. Wash pellet three times with 1 ml co-immunoprecipitation buffer. For each wash,
gently invert tube three times before pelleting. After each pelleting, use a 20-ml
syringe with an 18-G needle to aspirate and remove supernatant.
It may be possible to omit the costly protease inhibitors from the buffer at this stage.

8. Aspirate as much liquid as possible from the final without touching the beads and
add 25 l of 2 sample buffer.
If desired, samples containing sample buffer can be frozen up to several months at 80 C
prior to SDS-PAGE. In this case the buffer should be prepared with sterile stock solutions
and made with 1 mM sodium azide included.

9. Prepare for SDS-PAGE analysis by boiling for 5 min, vortexing, and microcen-
trifuging briefly to pellet beads. Use a Hamilton syringe to load eluates onto an
SDS-polyacrylamide gel, arranging duplicate samples to allow preparation of dupli-
cate blots. Separate by electrophoresis (UNIT 10.2A).
A Hamilton syringe works well to remove the eluate from the beads during loading.

10. Immunoblot duplicate samples separately with antibodies for each of the two proteins
(UNIT 10.8). Be sure to include aliquots of the whole-cell extract for comparison and
as a positive control for the immunoblot.
Each immunoblot can be reprobed with the antibody to the other protein.

ALTERNATE COPRECIPITATING A GST FUSION PROTEIN

PROTOCOL
GST fusion proteins may be coprecipitated by following the co-immunoprecipitation
procedure (see Basic Protocol) with the modifications outlined below. This procedure
might be used when the protein in question comigrates with immunoglobulin heavy or
light chain in an SDS-PAGE gel or if the antibodies being used precipitate too many
cross-reacting proteins, such as the protein being tested for association. Furthermore, it
is possible to dissociate the purified GST fusion from the solid-state glutathione resin
under gentle conditions through the use of imidazole. The approach is also useful in that
it will increase the size of the protein sufficiently that this size increase can be used as a
diagnostic feature in analyzing complexes.

Additional Materials (also see Basic Protocol)

Glutathione-agarose or glutathione-Sepharose slurry (see recipe)
1. Prepare duplicate samples as described for protein A/GSepharose (see Basic Pro-
tocol, step 1), omitting antibody.
2. Microcentrifuge 10 min at maximum speed, 4 C, to pellet nonspecific aggregates.
Transfer supernatant to new microcentrifuge tube.
3. Add 30 l glutathione-agarose or glutathione-Sepharose slurry (25- to 30-l bead
volume). Be sure to evenly suspend the slurry before distributing it to the samples.
4. Rotate the sample, pellet and wash glutathione-agarose/Sepharose, and perform
SDS-PAGE and immunoblot analysis (see Basic Protocol, steps 5 to 10).

Detection of
Protein-Protein
Interactions by
Coprecipitation

20.5.6
Supplement 76 Current Protocols in Molecular Biology
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see APPENDIX 4.

Co-immunoprecipitation buffer
50 mM TrisCl, pH 7.5 (APPENDIX 2)
15 mM EGTA
100 mM NaCl
0.1% (w/v) Triton X-100
Store at 4 C
Immediately before use add:
1 protease inhibitor mix (see recipe)
1 mM dithiothreitol (DTT)
1 mM phenylmethylsulfonyl fluoride (PMSF; from fresh 250 mM solution in 95%
ethanol)
If necessary for activity of the protein of interest, a divalent cation may need to be included
in the buffer and EGTA omitted (as for extraction buffer; see Strategic Planning).
The protease inhibitor mix, PMSF, and DTT should be added fresh at the time of experi-
mentation. The mixture without those components can be stored for months at 4 C with the
addition of 1 mM sodium azide. PMSF is labile in aqueous buffer and should be added at
the last minute. Protease inhibitor cocktails are also commercially available.
Since many protein-protein interactions are dependent upon phosphorylation, it may
be necessary to include phosphatase inhibitors in the whole-cell extracts and the co-
immunoprecipitation buffer phosphatase inhibitor solutions. A starting mix is 0.5 mM
vanadate (0.25 mM each meta- and ortho-vanadate or 0.5 mM sodium vanadate, pH 7.4),
10 mM sodium fluoride (NaF), and 10 mM -glycerol phosphate.

Glutathione-agarose or glutathione-Sepharose slurry

Swell 1.5 g glutathione-agarose or glutathione-Sepharose beads (e.g., Pierce,
Sigma) in 30 ml of 50 mM TrisCl, pH 7.5 (APPENDIX 2), for 1 to 2 hr on ice.
Pellet beads by gravity or very gentle centrifugation (1 min at 1000 rpm in a
tabletop centrifuge) and then wash four times with co-immunoprecipitation buffer
(see recipe) that lacks protease inhibitor mix and contains 1 mM sodium azide.
Resuspend beads in 15 ml of this buffer to yield a final slurry concentration of
100 mg/ml. Store at 4 C (stable for months).
This recipe can be scaled up or down.

Protease inhibitor mix, 1000

Dissolve in DMSO:
5 mg/ml chymostatin
5 mg/ml pepstatin A
5 mg/ml leupeptin
5 mg/ml antipain
Store in aliquots up to 1 year at 20 C
Protein A/GSepharose slurry
Swell 1.5 g protein A or protein GSepharose beads (e.g., Pierce, Sigma) in 30 ml
of 50 mM TrisCl, pH 7.5 (APPENDIX 2), for 1 to 2 hr on ice. Pellet beads by gravity or
very gentle centrifugation (1 min at 1000 rpm in a tabletop centrifuge) and then wash
four times with co-immunoprecipitation buffer (see recipe) that lacks protease in-
hibitor mix and contains 1 mM sodium azide. Resuspend beads in 15 ml of this buffer
Analysis of
to yield a final slurry concentration of 100 mg/ml. Store at 4 C (stable for months). Protein
Interactions
This recipe can be scaled up or down.
20.5.7
Current Protocols in Molecular Biology Supplement 76
 COMMENTARY
Background Information
Coprecipitation is a powerful and simple
approach to test for a physical interaction be-
tween proteins. There are many reasons to in-
corporate coprecipitation into a study. First,
as a form of protein affinity chromatography,
the method may be sensitive enough to de-
tect weak associations that do not withstand
the rigors of standard purification methods in-
volving substantial dilution of the initial cell
extract. Second, coprecipitation tests for as-
sociations between proteins within the milieu
of a whole-cell extract, where the proteins are
present at native concentration in a complex
mixture of other cellular components. This
feature makes it an important partner to two-
hybrid methods and direct tests of interactions
using purified proteins, because it provides a
way to verify that a positive interaction re-
flects a true in vivo association. For example,
nonphysiological interactions can be detected
when purified proteins are present at too el-
evated a concentration. A falsely positive in-
teraction between two proteins can also arise
in a two-hybrid test when protein domains are
inappropriately exposed due to altered fold-
ing. In addition, not all proteins are amenable
to two-hybrid analysis; a negative result may
mask a true association.
Nevertheless, a word of caution is in order.
The ability to coprecipitate two proteins from
a cellular extract is not proof that a particular
interaction normally takes place in vivo. Ad-
ditional experiments are needed to argue that
a given interaction is not the result of mix-
ing cell contents during extract preparation.
Such evidence could include colocalization of
the proteins or demonstration of functional
relatedness.
When performing coprecipitation, it is im-
portant to precipitate from both directions (i.e.,
individually precipitating protein 1 and protein
2, and testing for the presence of protein 2 and
protein 1, respectively). This is important in
that it can provide further verification of an
interaction between the proteins. It is also im-
portant because it is possible the interaction
will only be detected in one direction. An in-
ability to detect an interaction in one direction
could be due to a variety of factors including
obstruction of an interaction by the binding
of the antibody or other affinity agent, or dif-
ferences in pool size representation of each
Detection of protein. For example, protein 1 may bind to
Protein-Protein
Interactions by many proteins besides protein 2, while most
Coprecipitation of protein 2 binds to protein 1. In this sce-
20.5.8
Supplement 76
nario, it would be anticipated that detection of
their association will be most efficient when
protein 2 is precipitated.
Critical Parameters and
Troubleshooting
Two important factors in determining how
well an immunoprecipitation will work are the
affinity of the antibody for the antigenic site
on the protein (or the affinity of the protein tag
for the affinity matrix) and the yield of the rel-
evant proteins in the extract. For efficient im-
munoprecipitation, the antibody should have
an affinity to the antigen in the range of 107 to
109 M1 (Harlow and Lane, 1998). The eas-
iest way to enhance detection of the immune
complex is to increase the concentration of an-
tibody and the protein being immunoprecipi-
tated, as long as the concentration of antibody
is not already saturating. This can be deter-
mined empirically. A starting point is 1 g
antibody (Harlow and Lane, 1998), which is
typically 0.5 to 1 l of a polyclonal antiserum,
50 l of a hybridoma culture supernatant, or
0.5 ml of ascites fluid. The reaction volume
of the immunoprecipitation can be reduced
to increase the concentration of the protein
along with optimization of the extracts (see
below). With protein tagging, it is possible to
increase the number of copies of the moiety
being bound by affinity matrix as a means of
improving detection.
It is important to vary conditions of both
the extract preparation and the coprecipitation
to determine the optimal conditions. When
starting from scratch, it is most prudent to
use a range of lysis and precipitation con-
ditions ranging from less to more stringent
in terms of the amount of salt and non-
ionic detergent to determine which conditions
give the best yield of the desired proteins.
When no interaction is detected, it is worth-
while to use less stringent conditions (reduced
salt and nonionic detergent). In addition, it
may be necessary to avoid any dilution of
the whole-cell extract. This can be done by
adding the protein A/GSepharose directly to
the extract after an initial clarification centrifu-
gation and by using smaller wash volumes.
Depending on the strength and nature of the in-
teraction, the precipitation can be performed in
the presence of a mixture of detergents that in-
cludes ionic detergents (for example, 1% Tri-
ton X-100, 0.5% deoxycholate, 0.1% SDS).
In addition, it may be necessary to increase
the expression levels of the proteins in question
Current Protocols in Molecular Biology
in order to detect them readily by coprecipi-
tation. A range of expression levels is recom-
mended, because a level that is too high can
lead to unregulated interactions (Feng et al.,
1998). One can scale up the coprecipitation
to at least 2 mg of whole-cell extract in the
described assay (Wang et al., 2005); a start-
ing point is 0.25 to 1 mg. Here, the limit-
ing factor is the concentration of the extracts,
which must be high enough to allow the vol-
ume of the coprecipitation mixture to remain
low. Larger-scale extract preparations may be
necessary to generate more concentrated ex-
tracts. Finally, in cases of failure due to low
abundance of the proteins in the host organ-
ism, one can overexpress a tagged version of
one of the two proteins in the same or another
host (such as E. coli), concentrate this pro-
tein by pre-immobilization on an appropriate
affinity matrix, and then incubate the affixed
protein with extracts from the host organism.
The most important objective in these
experiments is to generate as great a
signal-to-noise ratio as possible and avoid
problems of background. A variety of pa-
rameters can be changed to enhance the co-
immunoprecipitation. Optimization of the pre-
cipitating antibody is one possibility. Protein
ASepharose and protein GSepharose should
give results comparable to anti-Ig serum.
However, direct coupling of the antibody to
Sepharose may lead to reduced background
and more quantitative precipitation. In addi-
tion, varying the ratio of antibody to whole-
cell extract and the total amount of whole-cell
extract is strongly suggested to determine the
optimal amount of antibody that gives the most
precipitation with the least amount of back-
ground. Affinity purification of the antibody
may be necessary if the antibody immunopre-
cipitates additional cross-reacting proteins.
Additional approaches can be taken to min-
imize background. First, better clarification of
the cell extract can be achieved by precen-
trifugation at 100,000 g. These extracts can
be directly used for coprecipitation without an
intervening freezing step, which can increase
the amount of protein precipitation. Second,
both the lysis buffer and the coprecipitation
buffer can be supplemented with 1% BSA
to reduce nonspecific binding to the affinity
matrix. Third, the whole-cell extract can be
preincubated with protein A/GSepharose to
remove nonspecific proteins that bind to the
solid support. Fourth, the amount of salt and
detergent can be increased in both the copre-
cipitation and the washes to reduce nonspe-
cific binding. Fifth, increasing the number of
Current Protocols in Molecular Biology
washes may also help, although it may reduce
the amount of specific protein that remains as-
sociated. Sixth, the expression levels of the
proteins in question can be increased to gener-
ate a stronger signal that is above background
binding. Alternatively, it may be possible to
produce a whole-cell extract that is enriched
for the proteins in question (e.g., by preparing
a nuclear extract if the proteins are known to be
in the nucleus). In instances where one of the
proteins binds nonspecifically to Sepharose,
the substitution of an agarose-based affinity
matrix may help solve the problem. Finally, it
may be necessary to generate a different set of
reagents to precipitate the proteins in question
(i.e., different antibodies and/or protein tag).
Anticipated Results
Provided suitable antibodies are available
to the proteins in question and the physical
interaction is stable under the coprecipitation
conditions, it should be possible to detect an
interaction between two proteins. This method
can be scaled up to coprecipitate sufficient pro-
tein to allow detection in a polyacrylamide gel
either by silver stain (1 to 10 ng/band) or
Coomassie blue stain (0.1 to 1 mg/band),
with subsequent excision of the band from the
gel and identification by mass spectrometry.
Time Considerations
Once the extracts are prepared, coprecip-
itation can be done within 3 to 4 hr, yield-
ing samples ready to load on a gel for SDS-
PAGE and immunoblot analysis. It is possible
to freeze the prepared extracts and thaw them
on ice prior to setting up the coprecipitation,
but higher yields may be obtained with sam-
ples that have not been frozen.
Literature Cited
Auerbach, D., Galeuchet-Schenk, B., Hottiger,
M.O., and Stagliar, I. 2002. Genetic approaches
to the identification of interactions between
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Transduct. Res. 22:471-481.
BioSupplyNet Source Book. 2005. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor,
N.Y.
Bornhorst, J.A. and Falke, J.J. 2000. Purification of
proteins using polyhistidine affinity tags. Meth-
ods Enzymol. 326:245-254.
Feng, Y., Song, L.-Y., Kincaid, E., Mahanty, S.K.,
and Elion, E.A. 1998. Functional binding be-
tween G and the LIM domain of Step is re-
quired to activate the MEKK Ste11. Curr. Biol.
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Field, J., Nikawa, J., Broek, D., MacDonald,
B., Rodgers, L., Wilson, I.A., Lerner, R.A., Analysis of
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and Wigler, M. 1988. Purification of RAS- Interactions
responsive adenylyl cyclase complex from
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Supplement 76
 Saccharomyces cerevisiae by use of an epitope
addition method. Mol. Cell. Biol. 8:2159-2165.
Gavin, A. and Superti-Furga, G. 2003. Protein com-
plexes and proteome organization from yeast to
man. Curr. Opin. Chem. Biol. 7:21-27.
Harlow, E. and Lane, D. 1988. Antibodies: A Labo-
ratory Manual. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.
Harlow, E. and Lane, D. 1998. Using Antibodies:
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Contributed by Elaine A. Elion
Harvard Medical School
Boston, Massachusetts
Current Protocols in Molecular Biology