FeatureArticle

3x FLAG:
Ultra-Sensitive Detection A.
Molecular
Biology

of Recombinant Proteins
by Ron Hernan, Ken Heuermann and Bill Brizzard
Sigma-Aldrich Corporation, St. Louis, MO, USA

Introduction
Epitope tagging has become a powerful tool for the
detection and purification of expressed proteins. This
methodology has been used for protein localization,
immunoprecipitation, and protein-protein interaction.
Many types of tags have been used, with c-myc and
FLAG® being two of the most popular epitope tags
utilized.1 Generally, these sequences are fused to the N- B.
or C-terminus of the expressed protein making them more
accessible for antibody detection and less likely to cause
severe structural or functional perturbations.

The original FLAG sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-

Lys, is recognized by two monoclonal antibodies, M1 and
M2.2,3 In addition, the FLAG sequence with an initiator
methionine attached is recognized by the M2 antibody
and a third antibody, M5.4 The last five amino acids of the
FLAG sequence are the recognition site for the protease
enterokinase, thus, allowing for removal of the FLAG
epitope tag.

The FLAG tag has been used in expression systems for

detection and purification of heterologous proteins in
E. coli,5 Saccharomyces cerevisiae,3,6 Drosophila,7 Figure 1. p3xFLAG-CMV-7 expression vector. (A) Plasmid map of the
p3xFLAG-CMV-7 showing the CMV promoter, human growth hormone
Baculovirus,8,9 and mammalian systems.10,11 For transcription termination and polyadenylation site, SV40 origin of replication,
mammalian expression systems, expression levels are low Col E1 origin of replication, and ß-lactamase gene. (B) DNA and protein
sequence of 3xFLAG-CMV-7 multiple cloning site and FLAG sequences.
and effective detection of expressed foreign proteins using
established methods can be difficult. We describe a
mammalian expression plasmid containing multiple FLAG
epitopes in tandem, p3x FLAG CMV-7, designed for
intracellular expression with increased sensitivity of
detection. This vector contains the cytomegalovirus (CMV)
promoter12 and simian virus 40 (SV40) origin of replication
for efficient expression in COS-7 cells.13 We compare the
detection of triple FLAG-tagged bacterial alkaline
phosphatase (BAP) expressed and purified from E. coli to
single FLAG-tagged BAP. We demonstrate the efficacy of
pFLAG-CMV-7 as a mammalian expression vector.

Materials and Methods

All materials were supplied by Sigma Chemical (St. Louis, MO)
unless otherwise stated.

Vector Construction Figure 2. Expression vector p3xFLAG-ATS-BAP. Vector map of p3xFLAG-

We have constructed a vector for expression of proteins in
mammalian host cells using a modified version of the
FLAG expression system, which contains 3x FLAG
sequences in tandem (Figure 1).
Expression of Bacterial
Alkaline Phosphatase in E. coli
To address whether a triple FLAG fusion protein produces
a more sensitive response than the traditional FLAG
ATS-BAP showing insertion of the phoA coding region into
pFLAG-ATS-BAP.
epitope tag, a triple FLAG version of bacterial alkaline
phosphatase was constructed for expression in E. coli
(Figure 2). The vector p3x FLAG-ATS-BAP was transformed
into E. coli and the 3x FLAG-BAP protein was expressed
and purified. In addition, an N-FLAG-BAP protein
containing the traditional epitope tag was also expressed
and purified.5

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Western Blot
Purified 3x FLAG-BAP and N-FLAG-BAP were diluted with
10
2x Laemlli buffer, boiled for five minutes and then placed
on ice. Samples were resolved on a 15% SDS-PAGE using
the method of Laemlli10 and then transferred to
nitrocellulose membranes. The membrane was blocked
with phosphate buffered saline containing 3% non-fat dry
milk for 1 hour and then rinsed three times in TBS, 0.05%
Tween® 20 (TBS-T). The membrane was incubated with
M2 antibody at a final concentration of 10 µg/ml for 30
minutes in TBS-T and then rinsed three times in TBS-T.
The membrane was then incubated for 30 minutes with a
goat anti-rabbit IgG (whole molecule) horseradish
peroxidase (HRP) conjugate, diluted 1:10,000 in TBS-T,
then rinsed three times in TBS-T. The FLAG-tagged
proteins were detected with the HRP conjugate and
visualized by chemilumenescent detection using ECL™ kit
(Amersham Pharmacia Biotech, Piscataway, NJ ) with
exposures from 1-30 minutes on Kodak X-Omat® MR film
(Eastman Kodak, Rochester, NY).
Expression of Bacterial Alkaline Phosphatase
in Mammalian Cells
Functionality of the pFLAG-CMV-7 mammalian vector
was demonstrated by transfection of COS-7 cells with
pFLAG-CMV-7-BAP and detection of BAP expression by
immunostaining. At 48 hours post-transfection cells were
fixed with methanol:acetone, washed, and incubated for
1 hour with 10 µg/ml M2 monoclonal antibody:HRP
conjugate in TBS. Cells were then washed and stained
with 100 µg/ml o-dianisidine in the presence of 0.015%
hydrogen peroxide, in TBS. Cells not stained with
o-dianisidine were counter-stained for 1 minute using a
1:1 solution of Mayer's hematoxylin in water.
Results
This vector construct was designed to improve the
detection limit of expressed proteins in mammalian host
cells. The first two flag FLAG peptides are modifications
of the original FLAG sequences previously described: Asp-
Tyr-Lys-Asp-His-Asp. A Gly-Ile spacer joins the sequences.
These alternative sequences arise from phage display
studies in which a different binding motif was
determined.14 This allows the introduction of additional
FLAG antibody binding sites without the addition of extra
enterokinase recognition/cleavage sites.
The p3x FLAG-CMV-7 expression vector contains the
promoter region of the human cytomegalovirus major
immediate early gene, which allows for constitutive
expression of cloned genes in mammalian cell lines. The
Kozak consensus sequence15 is provided in the vector
along with a multiple cloning site, which allows for a
variety of cloning strategies. The multiple cloning site is
compatible with the other existing CMV mammalian
expression vectors. In addition, the expression vector
contains the SV40 origin of replication for efficient high-
level transient expression13 and a DNA segment from the
human growth hormone containing transcriptional
termination sequence and polyadenylation signals.16
p3x FLAG-CMV-7 contains the ß-lactamase gene for
selection of the plasmid in E. coli. Using this vector, we
have successfully transfected and expressed heterologous
proteins in COS cells.
Bacterial Alkaline Phosphatase Expression
Comparison of the sensitivity of the single FLAG-BAP
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1 2 3 4 5
A. 3x FLAG BAP
Molecular
Biology
B. 1x FLAG BAP
Figure 3. Detection of purified 3xFLAG-BAP by Western blot. (A) Western
blot of purified 3xFLAG-BAP using ANTI-FLAG M2 antibody (Lane 1, 0.5 ng;
Lane 2, 1.0 ng; Lane 3, 2.0 ng; Lane 4, 5.0 ng; and Lane 5, 10 ng). (B) Western
blot of purified N-FLAG-BAP using ANTI-FLAG M2 antibody (Lane 1, 0.5 ng;
Lane 2, 1.0 ng; Lane 3, 2.0 ng; Lane 4, 5.0 ng; and Lane 5, 10 ng).
A Figure 4. Light
microscopy of
immunostained cells.
(A) COS-7 cells transfected
with p3xFLAG-CMV-7-BAP.
(B) Control COS-7 cells.
B
versus the triple FLAG-BAP was demonstrated by Western
blot analysis as previously described. Figure 3 shows the
Western blot of purified single FLAG-BAP and triple flag
FLAG-BAP probed with ANTI-FLAG®-M2 monoclonal
antibody and detected by chemiluminescence. The results
clearly indicate a 10-fold increase in detection limit of the
triple FLAG-BAP compared to the single FLAG-BAP fusion
protein. We were able to detect 500 picograms of
purified 3x FLAG- BAP with exposures as short as
1 minute. With increased exposure time, detection as low
as 100 picograms has been achieved but with increased
background (data not shown).
COS-7 cells expressing FLAG-tagged BAP fusion protein
are shown in Figures 4(A) and (B). Detection of
transfected cells is typically observed within 10 minutes of
adding o-dianisidine.
Discussion
The FLAG epitope tag has been effectively used to detect
and purify proteins5 in mammalian and bacterial systems.
We have demonstrated that the presence of three FLAG
epitopes greatly increases the detection limit of purified
bacterial alkaline phosphatase. Moreover, we have found
that 3x FLAG-BAP cannot be eluted from ANTI-FLAG M2
affinity gel by competition with the original FLAG peptide.
However, 3x FLAG-BAP and the 1x FLAG-BAP can be
competitively eluted from the ANTI-FLAG M2 affinity gel
using 3x FLAG peptide (data not shown). The
p3x FLAG-CMV-7 vector was designed for expression
and detection of heterologous proteins in mammalian cells
and is compatible with existing pFLAG-CMV vectors. This
allows for easy subcloning between vectors containing the
single FLAG and the triple FLAG. The immunostaining
results show that expression of the phoA gene, which
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w w w. s i g m a - a l d r i c h . c o m
 codes for BAP in COS-7 cells, is not significantly perturbed 7. Xu, T., and Rubin, G. Analysis of genetic mosaics in developing and adult
Drosophila tissues. Development, 117, 1223-1237 (1993).
by addition of the 3x FLAG sequence.
8. Dent, P., et al., Regulation of raf-1 and raf-1 mutants by ras-dependent and
ras-independent mechanisms in vitro. Mol.Cell Biol., 15, 4125-4135 (1995).
The M2 antibody reacts with the alternate epitope in the
3x FLAG sequence. In contrast, M5 antibody fails to show 9. Ritchie, P., et al. Baculovirus expression and biochemical characterization of
the human microsomal triglyceride transfer protein. Biochem. J., 338, 305-310
the increased sensitivity that the M2 antibody (1999).
demonstrates, (results not shown). Recent results using
Molecular
Biology

phage display17 have demonstrated that the critical
residues for M2 binding and M5 binding are slightly
different. M2 antibody prefers the sequence Asp-Tyr-Lys-
Xxx-Xxx-Asp-Xxx-Xxx, while M5 prefers Asp-Tyr-Xxx-Xxx-
Asp-Asp-Xxx-Xxx. The triple FLAG sequence Asp-Tyr-Lys-
Asp-His-Asp clearly favors the binding of M2 over that of
M5 or M1 antibodies. This expression system allows for
increased sensitivity and detection of the FLAG epitope
tagging system while retaining the benefits of the FLAG
mammalian expression system. These include FLAG’s
hydrophilic nature, small size, and the ability to remove
the FLAG tag using enterokinase.
10. Schulte am Esch II, J. et al., Structural elements and limited proteolysis
of CD39 influence ATP diphosphohydrolase activity. Biochemistry, 38,
2248-2258 (1999).
11. Overholt, S., et al., Head and neck squamous cell growth suppression
using adenovirus-p53-FLAG: a potential marker for gene therapy trials.
Clin.Cancer Res., 3, 185-191 (1997).
12. Thomsen, D., et al., Promoter-regulatory region of the major immediate
early gene of human cytomegalovirus. Proc. Natl. Acad. Sci. USA, 81,
659-663 (1984).
13. Okayama, H., and Berg, P., A cDNA cloning vector that permits expression
of cDNA inserts in mammalian cells. Mol. Cell. Biol., 3, 280-289 (1983).
14. Miceli, R., et al., Two-stage selection of sequences from a random phage
display library delineates both core residues and permitted structural range
within an epitope. J. Immunol. Methods, 167, 279-287 (1994).

References
1. Evan, G., et al., Isolation of monoclonal antibodies specific for human
c-myc proto-oncogene product. Mol. Cell. Biol., 5, 3610-3616 (1985).
2. Hopp, T., et al., A short polypeptide marker sequence useful for
recombinant protein identification and purification. BioTechnology, 6,
1204-1210 (1988).
3. Prickett, K., et al., A calcium dependent antibody for identification and
purification of recombinant proteins. BioTechniques, 7, 580-589 (1989).
4. Brizzard, B., and Chubet, R., in Current Protocols in Neuroscience. Crawley,
J. N., et al., Eds, pp. 5.8.1-5.8.11 (John Wiley & Sons, New York, N.Y., 1999).
15. Kozak, M., Point mutations close to the AUG initiator codon affect the
efficiency of translation of rat preproinsulin in vivo. Nature, 308, 241-246
(1984).
16. Seeburg, P., The human growth hormone gene family: nucleotide
sequences show recent divergence and predict a new polypeptide hormone.
DNA, 1, 239-249 (1982).
17. Slootstra, J., et al., Identification of new tag sequences with differential
and selective recognition properties for the anti-FLAG monoclonal M1, M2
and M5. Molecular Diversity, 2, 156-164 (1996).
Adapted from Biotechniques 28, 789-793 (April 2000) by
permission. Figures 1 (p. 790) and 2 (p. 791), adapted.

5. Brizzard, B., et al. Immunoaffinity purification of FLAG epitope-tagged
bacterial alkaline phosphatase using a novel monoclonal antibody and peptide
elution. BioTechniques, 16, 730-735 (1984).
6. Lee, J., et al. A protein kinase involved in the regulation of inflammatory
cytokine biosynthesis. Nature, 372, 739-746 (1994).
FLAG and ANTI-FLAG are registered trademarks of the Sigma-Aldrich
Corporation. Tween is a registered trademark of ICI. ECL is a trademark
of Amersham Pharmacia Biotech. X-Omat is a registered trademark of
Eastman Kodak.

About the Authors

Ron Hernan, Ph.D., and Ken Heuermann, M.S., are senior scientists in Recombinant Protein Expression R&D at Sigma-Aldrich,
St. Louis, MO. Bill Brizzard, Ph.D., is the manager of Technology Transfer at Sigma-Aldrich, St. Louis, MO.

ORDERING INFORMATION
Product Code Product Name Unit Price
E 2400 p3x FLAG-CMV™-7 Expression Vector 20 µg $238.60
E 4026 p3x FLAG-CMV™-7.1 Expression Vector 20 µg $250.00
E 4151 p3x FLAG-CMV™-8 Expression Vector 20 µg $250.00
E 4276 p3x FLAG-CMV™-9 Expression Vector 20 µg $250.00
E 4401 p3x FLAG-CMV™-10 Expression Vector 20 µg $250.00
E 4776 p3x FLAG-CMV™-13 Expression Vector 20 µg $250.00
E 4901 p3x FLAG-CMV™-14 Expression Vector 20 µg $250.00
*All p3xFLAG vectors include N-terminal p3xFLAG-CMV-BAP as control.
F 4799 3x FLAG peptide 4 mg $152.30
25 mg $710.50
P 2104 N-TERMINAL 3x FLAG-BAP 0.1 mg $130.00

RELATED PRODUCTS
Product Code Product Name Unit Price
A 1205 ANTI-FLAG M2 Affinity Gel 1 ml $227.90
5 ml $481.00
10 ml $961.90
25 ml 1359.70
F 3165 ANTI-FLAG M2 Antibody 0.2 mg $136.30
1 mg $232.80
5 mg $384.00

SUPPORTING LITERATURE
Recombinant Protein Expression booklet (BQW)

WEB SITE LINKS

http://www.sigma-aldrich.com/saws.nsf/SigProducts?OpenFrameset

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