TransLISA, a novel quantitative, nonradioactive assay
for transcription factor DNA-binding analyses
Kristiina A. Vuori
1
, Johanna K. Ahlskog
2
, Lea Sistonen
2
and Mikko Nikinmaa
1
1 Centre of Excellence in Evolutionary Genetics and Physiology, Department of Biology, University of Turku, Finland
2 Department of Biology, A
˚
bo Akademi University and Turku Centre for Biotechnology, University of Turku and A
˚
bo Akademi University,
Finland
Introduction
Transcription factors are proteins that bind DNA to
induce or suppress gene transcription. They function
in virtually all biological processes, although their
role in transcriptional regulation in eukaryotes is
poorly understood [1]. Among the most intensively
studied transcription factors is heat shock factor 1
(HSF1). HSF1 binding to its response elements in
target gene promoters is an established model system
of inductive transcriptional regulatory responses, and
studies on HSF1 have yielded important insights into
basic cellular and molecular biology and contributed
to drug discovery [2–4]. As transcriptional regulation
involving specific transcription factors has both basic

tion factors in cell and tissue lysates. TransLISA outperforms EMSAs,
because it eliminates the need to use radioactive chemicals and allows fast
and precise quantification of DNA-binding activity of transcription factors
from large number of samples simultaneously. We have used TransLISA to
demonstrate the DNA-binding activity of heat shock factor 1, representing
a well-known model of inductive transcriptional regulatory responses, but
the method is easily adaptable for the study of any transcription factor.
Thus, TransLISA can replace EMSAs and may be used in various applica-
tions and research fields where quantitative, cost-effective and large-scale
measurements of the DNA-binding activity of transcription factors are
required, including screening of responses in multiple treatments in cellular
and molecular biology, evolutionary research, environmental monitoring,
and drug discovery.
Abbreviations
CV, coefficient of variation; EMSA, electrophoretic mobility shift assay; HSE, heat shock element; HSF1, heat shock factor 1; Hsp, heat
shock protein; LOCI, luminescent oxygen channeling immunoassay; MEF, mouse embryonic fibroblast.
7366 FEBS Journal 276 (2009) 7366–7374 ª 2009 The Authors Journal compilation ª 2009 FEBS
Currently, DNA-binding activities of transcription
factors are generally analyzed with electrophoretic
mobility shift assays (EMSAs) [5,6]. Cell or tissue
extracts are mixed with a radiolabeled oligonucleotide
probe containing the binding site for the transcription
factor of interest. Binding reactions are run in non-
denaturating polyacrylamide gels (PAGE). Gels are
dried and exposed to X-ray film overnight or longer
(Fig. 1A). The intensity of the resulting bands, gener-
ally 15 per gel at most, can be quantified with imaging
software. However, EMSA is time-consuming, does
not allow high-throughput analysis, and provides only
descriptive or semiquantitative results. In addition, it

PAGE
Pipet to plate, add A beads and incubate
+16 h
D
1 h
Autoradiography
Add D beads and incubate
D A
O
2
Read AlphaLISA si
g
nal at 615 nm
HSE
Fig. 1. Comparison of EMSA and TransLISA for the detection of HSF1–DNA binding activity. (A) Schematic presentation of EMSA assay.
Cell or tissue extracts or peptides are incubated with radioactively labeled (c
32
P) probe containing HSF1-binding sites. Binding reactions are
run in nondenaturating polyacrylamide gel. Gels are dried and exposed to X-ray film overnight or longer. The amounts of DNA-bound HSF1
complexes in the samples are detected by band intensities in the autoradiograph. (B) Schematic presentation of TransLISA. Cell or tissue
extracts or peptides are incubated with biotin-labeled probe containing HSE. Aliquots of binding reactions are pipetted into a 384-well plate,
and acceptor beads containing antibody against HSF1 are added to the wells. After incubation, streptavidin-coated donor beads are added,
and plates are covered and incubated at room temperature in the dark. When the acceptor beads are brought into proximity to the donor
beads via HSF1–DNA interactions, singlet oxygen generated by excitation at 680 nm initiates a series of luminescent energy transfers
between compounds in the acceptor beads. The resulting emission is read at 615 nm with a plate reader.
K. A. Vuori et al. TransLISA
FEBS Journal 276 (2009) 7366–7374 ª 2009 The Authors Journal compilation ª 2009 FEBS 7367
DNA binding in biological samples. Our assay is based
on the no-wash ELISA platform AlphaLISA (ampli-
fied luminescence proximity homogeneous) (Perkin-

transcription factors, with one HSF in yeast, nematode
worms and fruit flies, and four members, HSF1–HSF4,
in vertebrates. HSF1 is required for the heat shock
response, which is triggered by proteotoxic stressors
such as elevated temperature and heavy metals. Upon
activation, HSF1 trimerizes, undergoes hyperphosph-
orylation, and binds to heat shock elements (HSEs) in
the promoters of heat shock genes, which code for
heat shock proteins (Hsps), molecular chaperones that
facilitate correct folding of nascent and misfolded
proteins [2].
Here, we describe the development and performance
of a 384-well plate immunoassay, named TransLISA,
for measuring the DNA-binding activity of a transcrip-
tion factor (Fig. 1B). This assay is the first homoge-
neous, nonradioactive assay for rapid and sensitive
quantification of the DNA binding of transcription
factors in cell and tissue lysates. Although we have
developed the assay using HSF1, it can easily be
adapted for the study of any transcription factor. The
method can thus replace EMSA whenever oligonucleo-
tides containing response elements and specific anti-
bodies for the transcription factor are available. The
assay is the first assay suitable for high-throughput
measurements of transcription factor–DNA interac-
tions in biological samples. Therefore, it can be used
in various research applications, especially when mea-
surements of tens, hundreds or thousands of samples
are needed. Such applications may include screening of
cellular responses in multiple treatments, drug discov-

and heat-shocked samples (11.2-fold) was obtained
with 5 lg of total protein (Fig. 3A), and the greatest
difference between MEF control and heat-shocked
samples (17.4-fold) was achieved with 10 lg of total
protein (Fig. 3B). As the HeLa cell incubation reac-
tions with 5 and 10 lg of protein gave very similar
results, 11.2-fold or 10.4-fold difference, respectively,
between control and heat-shocked samples, 10 lgof
total protein in a 20 lL initial binding reaction was
selected for use in all assays.
TransLISA K. A. Vuori et al.
7368 FEBS Journal 276 (2009) 7366–7374 ª 2009 The Authors Journal compilation ª 2009 FEBS
We also tested the default assay protocol in which
2.5 lL of sample, 2.5 lL of probe and 10 lL of accep-
tor beads were first added to the plate wells and incu-
bated for 30 min at 4 °C, and this was followed by
addition of 10 lL of donor beads and 1 h of incuba-
tion at room temperature before reading. This type of
assay setup, however, resulted in only 4.1-fold and 6.3-
fold differences between HeLa cell and MEF control,
respectively, and heat-shocked samples (Fig. 3A,B).
Therefore, we concluded that the best resolution of the
assay is achieved by including the first 30 min incuba-
tion step, which allows the protein–DNA complexes to
form, as in EMSA.
Competition experiments
Competition experiments are used to confirm the speci-
ficity of the DNA-binding reaction in EMSA. In our
competition experiments, both unlabeled HSE probe
and blocking of the antibody with HSF1 peptide

HeLa
Control
Heat shock
0
5
10
15
20
25
30
35
10
Counts ×10
3
MEF
Control
Heat shock
0.01 0.1
nM Probe
0.01 0.1 1
nM Probe
A
B
Fig. 2. Optimization of probe concentrations. Increasing concentra-
tions (working concentration of 1–250 n
M; 0.05–12.5 nM in the incu-
bation reaction) of biotinylated oligomer probe were applied to
incubation reactions containing 10 lg of control and heat-shocked
HeLa cell (A) and MEF (B) protein extracts. Circles represent the
mean counts of triplicate wells, and the error bars represent stan-

tein extract amounts in the first incubation reaction were tested
with the HeLa cell (A) and MEF (B) control and heat-shocked sam-
ples by adding 1, 5 or 10 lg of protein extract to the reactions. In
addition, testing of the default protocol using the same samples
without the initial incubation step was included. The probe concen-
tration was the same (working concentration of 150 n
M; 0.75 nM in
the well) for all of the optimizations. The bars represent the mean
counts of triplicate wells, and the error bars represent standard
deviations of triplicate well counts.
K. A. Vuori et al. TransLISA
FEBS Journal 276 (2009) 7366–7374 ª 2009 The Authors Journal compilation ª 2009 FEBS 7369
DNA-binding activity (Fig. 5). The signal decreased in
50 and 500 nm recombinant HSF1 when compared to
the counts of 10 nm HSF1 peptide sample. This is due
to the ‘hook effect’, whereby the signal increases with
increasing target molecule concentration up to a cer-
tain point, after which the target molecule becomes
inhibitory in the reaction because of the saturation of
the available binding sites [16]. The dissociation con-
stant, K
d
, determined from the recombinant HSF1
DNA-binding experiment was 6.27 nm.
The sample-specific intraplate variability was
assessed by pipetting specific samples in different, ran-
domly selected positions within the plate. In eight of
11 cases, the total coefficient of variation (CV) value
of the wells was less than 10%, and in only one of 11
cases was the CV value of the wells unacceptably high

20
40
60
80
Counts ×10
3
Counts ×10
3
Counts ×10
3
0
0.1 1 10 100
0.01 0.1 1 10 100
Heat shock Mutated Scrambled
Fig. 4. Competition experiments. (A) Unlabeled HSE probe in the
incubation reaction abolished the signal of HeLa cell heat-shocked
sample in a dose-dependent manner. The units on the x-axes are
concentrations in the incubation reaction. The signal level of
untreated sample is indicated by the label ‘Heat shock’ on the graph.
(B) Blocking the antibody with recombinant HSF1 peptide abolished
the signal of HeLa cell heat-shocked sample in a dose-dependent
manner. The units on the x-axes are concentrations in the acceptor
bead preincubation reaction. The signal level of untreated sample is
indicated by the label ‘Heat shock’ on the graph. (C) Replacing the
correct HSE probe with mutated probe or with a nonsense ‘scram-
bled’ probe resulted in an absence of signal. The circles represent
the mean counts of triplicate wells, and the error bars represent
standard deviations of triplicate well counts.
Table 1. 5¢-Biotinylated and standard oligonucleotides used in
TransLISA development. The core DNA-binding sequences are indi-

known concentrations of recombinant human HSF1 protein instead
of cell extract in a normal assay procedure. The circles represent
the mean counts of triplicate wells, and the error bars represent
standard deviations of triplicate well counts. The units on the x-axis
are concentrations in the incubation reaction.
TransLISA K. A. Vuori et al.
7370 FEBS Journal 276 (2009) 7366–7374 ª 2009 The Authors Journal compilation ª 2009 FEBS
specific signal (counts) is shown in the top panel of
Fig. 6. All of the results obtained from independent
assays were in line with each other, indicating good
reproducibility of the assay. The CV values of within-
assay triplicates for each sample on different days
(dots) and the percentage variation between different
assays (line) are shown in the bottom panel of Fig. 6.
The within-assay triplicate CV values were consistently
below 10%. The percentage interassay variation of sig-
nals (sample-specific CV values between assays run on
different days) was 8.3–15.1%, with the exception of
one sample, where the variation was 22.5%.
Measurements of biological samples
We measured three biological replicates of control,
heat-shocked and recovering HeLa cell and MEF sam-
ples (Fig. 7A,B). The results show, on average, 8.4-fold
induction of HSF1 DNA-binding in heat-shocked
HeLa cells, and, on average, 25.8-fold induction of
HSF1 DNA-binding in heat-shocked MEFs when
compared to the untreated cells. The results are well in
line with the results obtained from EMSA (representa-
tive images of HeLa cell and MEF control, heat shock
and recovery are shown in Fig. 7A,B, top left panels),

120
140
HeLa
60
70
80
90
MEF
C
A
B
HS R
CHSR
40
60
80
Counts ×10
3
30
40
50
Counts ×10
3
0
20
0
10
20
Control
Heat shock

after 0 to 40 (HeLa) or 0 to 60 (MEF) min of heat
shock (Fig. 8A,B). The results indicate a very fast
response in both HeLa cells and MEFs; the DNA-
binding activity of HSF1 increased markedly already
after 10 or 15 min when cells were exposed to heat
shock. These results agree with those of earlier studies
[17,18].
Discussion
In this study, we have established a 384-well plate,
nonradioactive, homogeneous immunoassay for quan-
tifying the DNA-binding activity of the transcription
factor HSF1 in cell extracts. In comparison with the
traditional method, EMSA, the novel TransLISA
assay is superior in many ways. This assay eliminates
the use of radioactivity and the need to run gels, and
gives results much more rapidly; also, the plate format
enables cost-effective high-throughput sample analysis.
The homogeneous assay format excludes the need for
any washing steps between the addition of reagents.
The broad analytical range of the assay allows quanti-
tation of large differences in the DNA-binding activity
of transcription factors. This is precluded in EMSA
analysis, owing to overexposure of the autoradiograph
when visualizing both strong and weak signals at the
same time. In addition, given the versatility of the
AlphaLISA platform, the detection of DNA-bound
transcription factors can easily be modified by using
different antibodies to either full-length or specific
epitopes of the protein, or to different tags. The anti-
bodies may be either directly coated on the acceptor

model. However, it is possible to establish TransLISA
assays for any transcription factor from any species
when the consensus binding sites are known and spe-
cific antibodies for the transcription factor are avail-
able. The assay described here may thus serve to
initiate further development of quantitative, cost-effec-
tive and large-scale measurements of the DNA binding
of transcription factors in biological samples, both in
basic research and drug discovery.
Experimental procedures
Cell culture, treatments and sample preparation
HeLa cells were cultured in DMEM supplemented with
10% fetal bovine serum, 2 mml-glutamine, penicillin, and
streptomycin, and MEFs were maintained in DMEM sup-
plemented with 10% fetal bovine serum, 1.2 mm sodium
pyruvate, l-glutamine, penicillin, and streptomycin. All cells
were maintained at 37 °C in a humidified 5% CO
2
atmo-
sphere. Heat shock treatment was performed in a 42 °C
(HeLa cells) or 43 °C (MEFs) water bath for the indicated
times. The recovery samples were heat-shocked for 1 h, and
then incubated at 37 °C for 3 h. Sample preparation and
EMSA were performed as described previously [5].
The protein contents of samples were determined with
the Bradford method, using the BioRad Protein Assay
(BioRad, Espoo, Finland) with BSA (Sigma-Aldrich,
St Louis, MO, USA) as the standard.
Assay components
Streptavidin-coated donor beads and protein A-coated

tion, 1–10 lg of protein extracts was incubated with 1 lLof
1–250 nm biotinylated oligonucleotide probe in a 20 lL reac-
tion in binding buffer (containing 10 mm Tris, pH 7.5,
50 mm NaCl, 4 mm EDTA, 20% glycerol), and 1 lgof
poly(dIdC) (Sigma-Aldrich, St Louis, MO, USA). After opti-
mization (see Results), 10 lg of protein and 150 nm probe
were selected for use in the consecutive assays. After the ini-
tial incubation step, 2 lL of protein extract ⁄ probe mix was
pipetted into the plate wells in triplicate, and 9 lL of protein
A acceptor beads (working concentration of 50 lgÆmL
)1
)
preincubated for 1 h with antibody against HSF1 (working
concentration of 2 lgÆmL
)1
) was added. The plates were cov-
ered and incubated at 4 °C in the dark for 30 min. Nine
microliters of streptavidin-coated donor beads (working con-
centration of 50 lgÆmL
)1
) was then added, and the plates
were covered and incubated at room temperature in the dark
for 1 h. The plates were read with an Envision Xcite instru-
ment (PerkinElmer Wallac, Turku, Finland). The final con-
centrations of the assay components in the wells were as
follows: probe, 0.75 nm; antibody, 0.8 lgÆmL
)1
; acceptor
and donor beads, both 20 lgÆmL
)1

of Turku (M. Nikinmaa and K. A. Vuori), the
Academy of Finland and A
˚
bo Akademi University
120
140
A
B
70
80
0102040
0153060
60
80
100
30
40
50
60
0
20
40
Counts ×10
3
0
10
20
0 102040
0153060
Counts ×10

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7374 FEBS Journal 276 (2009) 7366–7374 ª 2009 The Authors Journal compilation ª 2009 FEBS