Journal of Analytical Sciences, Methods and Instrumentation, 2013, 3, 173-178
http://dx.doi.org/10.4236/jasmi.2013.33022 Published Online September 2013 (http://www.scirp.org/journal/jasmi)
173
Determination of Interaction between NFκB p50 and
β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion
Muniasamy Neerathilingam1,2*, Sai Gandham2, Falguni Patel1, Mohammad Nasiruddin1
1Centre for Cellular Molecular Platforms (C-CAMP), NCBS-TIFR, GKVK Campus, Bangalore, India; 2Institute of Molecular Medi-
cine (IMM), University of Texas Health Science Center, Houston, USA.
Email: *munish@ncbs.res.in
Received July 28th, 2013; revised August 25th, 2013; accepted September 1st, 2013
Copyright © 2013 Muniasamy Neerathilingam et al. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ABSTRACT
NF-κB plays a crucial role in regulating various biological processes including innate and adaptive immunity, inflam-
mation, stress responses, B-cell development, and lymphoid organogenesis. Currently, several assays like electropho-
retic mobility shift assay (EMSA), enzyme-linked immunosorbent assay (ELISA), fluorescence resonance energy
transfer (FRET) and time-resolved fluorescence resonance energy transfer (TR-FRET) are widely used for studying the
NFκB intraction with β-IFN-κB binding oligo. Each of these techniques has varying utility with distinct strengths and
weaknesses. We describe a method AlphaLISA to identify NFκB p50 protein and β-IFN-κB binding oligo sequence and
interaction is efficient at a given concentration (10 nM) in the EMSA and Biacore’s SPR assays. The method has many
advantages such as use of small volume, high throughput (HTP), convenience of sample preparation and data analysis.
Keywords: DNA-Protein Interaction; Binding Constant; Equilibrium Constant; AlphaLISA; High Throughput (HTP)
1. Introduction
Protein-DNA interactions control the defining hallmarks
of all cellular processes and functions like DNA tran-
scription, packaging, replication and repair [1]. These
interactions can be studied using the classical steady-
state and time-resolved methods to understand the con-
formational changes and enzymatic reactions for a given
Protein-DNA complex [2]. In this study we have taken
the case of NF-κB.
NF-κB is a sequence specific transcriptional factor
made up of different protein dimers that binds to a com-
mon sequence motif known as the κB site. NF-κB plays a
key role in regulating a broad range of biological proc-
esses including innate and adaptive immunity, inflamma-
tion, stress responses, cell proliferation, apoptosis, mi-
gration, and lymphoid organogenesis. Disregulation of
NF-κB has been linked to inflammatory disorders, cancer,
autoimmune diseases and metabolic diseases [3-6]. Pri-
marily regulation of gene expression occurs at the level
of transcription [7]. IκB, the inhibitor and central regula-
tor of NFκB which prevents nuclear import of NFκB and
disrupts the NFκB-DNA complexes in the nucleus and
retain NFκB in cytoplasm. Proteasomal degradation of
IκB leads to the transport of NFκB into nucleaus where it
binds to DNA sites in its target promoters and regulate
transcription [8].
Currently, assays like electrophoretic mobility shift
assay (EMSA), enzyme-linked immunosorbent assay
(ELISA), fluorescence resonance energy transfer (FRET)
and time-resolved fluorescence resonance energy transfer
(TR-FRET) are widely used for studying DNA-protein
interactions. Each of these techniques has varying utility
with distinct strengths and weaknesses. For example,
EMSA is less informative in terms of protein-DNA in-
teraction dynamics and protein identity [9]. ELISA is
another broadly used method that provides high sensitiv-
ity, but needs cumbersome wash steps for antibody ad-
sorption owing to its larger well surface area. The draw-
backs of ELISA include high cost, labour-intensive and
low dynamic range [10]. TR-FRET and FRET assays
have a proximity detection limit of within 2 nm distance
[11]. Biacore Surface Plasmon Resonance (SPR) cannot
be used for high throughput (HTP) screening owing to
the need for multiple runs. The notable drawbacks of this
system are background resonance and limited range of
Kon and Koff values [12]. Despite the above limitations,
*Corresponding author.
Copyright © 2013 SciRes. JASMI
Determination of Interaction between NFκB p50 and β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion
174
many laboratories still employ these assays to study
DNA-protein interactions. Alpha (amplified luminescent
proximity homogeneous assay) technology is combined
with ELISA to create AlphaLISA—a generalised, yet
simple and homogenous bead-based assay used to study
biomolecular interactions in a highthroughput micro
plate format [13] and its well established protocols are
being utilized to study protein-protein and protein-ligand
interactions. Although, AlphaLISA assay is not a well-
established as the methods mentioned earlier, it is spe-
cifically designed to eliminate some of the drawbacks of
other conventional methods. AlphaLISA assays are less
cumbersome owing to the shorter incubation times, use
of significantly lower sample volume (usually 5 - 20 µl)
and miniaturization. The assay offers flexibility to use 96,
384, or 1536 well plates as needed. These are the key
factors that reduce the cost of screening while increasing
the throughput. AlphaLISA assays are versatile and can
act as an efficient method to identify the weak interac-
tions [13-15]. In addition to providing high-quality data
and NF-κB p50 protein and β-IFN-κB binding sequence
oligo interaction robust performances, the assays are
simple to set up and quick to optimize. The beads are
coated with a layer of hydrogel providing functional
groups forbio-conjugation. For example, streptavidin-
coated donor beads capture a biotinylated antibody
whereas the acceptor beads are conjugated with a secon-
dary antibody that recognizes different epitopes of the
analyte [14]. Various types of donor/acceptor beads are
available, thus providing flexibility in selection of beads
to study ligand-analyte binding interaction.
2. Materials and Methods
This study was designed to establish a defined protocol
for identifying the NFκB p50 protein and β-IFN-κB
binding oligo sequence interaction in a HTP fashion us-
ing AlphaLISA technique. We have demonstrated the
AlphaLISA protocol to study NFκB p50 protein binding
to a biotinylated homodimeric p50 binding DNA motif
(β-IFN-κB) (5’-TGGGAATTCCC-3’) (Supplementary
Figure S1). This biomolecular interaction system was
chosen based on previous reports that NFκB p50 protein
and β-IFN-κB DNA binds efficiently at a given concen-
tration in the EMSA and Biacore’s SPR assays [2,16-19].
In our study, the dynamic range for NFκB p50 protein
and β-IFN-κB binding assay was determined by prepar-
ing dilution series ranging from 0.1 nM to 100 nM con-
centration of both protein and oligo. Streptavidin-coated
donor beads were used since the DNA motif was bioti-
nylated and anti-GST conjugated acceptor beads for our
GST tagged protein [14] (Supplementary Figure S1).
Our protocol comprises of four steps: 1) Mix oligo and
protein, 2) Add the acceptor beads, 3) Add the donor
beads and 4) Measure chemiluminescence intensity of
samples (Supplementary section, and Figures S2-S5).
Detailed protocols are given in supplementary materials.
3. Results and Discussion
The data were generated using EnSpireTM Alphaplus
multilabel plate reader (PerkinElmer, USA). The binding
of NF-κB p50 protein and β-IFN-κB binding sequence
oligo was detected from the signal obtained from the
multiplate reader plotted against the DNA concentration
(Figures 1(a) and (b)). The assay was performed in a 96
half-well format with varying range of DNA (oligo) and
protein concentrations of 0.3 nM to 10 nM (Supplemen-
tary material) to determine the optimum concentration
for saturation of the functional sites on both beads. The
saturation curve of the chemiluminescence vs. DNA con-
centration showed an increase in rate of association with
(a)
(b)
Figure 1. a) Graph depicting the signal generated by En-
SpireTM Alphaplus reader against varying concentrations of
DNA and protein. The maximum peak was observed at 10
nm; b) Graph depicting the hooking point effect generated
by EnspireTM Alphaplus reader against varying concentra-
tions of DNA-protein. The binding equilibrium constant
“hooking point” was observed at 10 nm and then gradually
decreased with the increasing DNA and protein concentra-
tions.
Copyright © 2013 SciRes. JASMI
Determination of Interaction between NFκB p50 and β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion 175
increasing concentration of both oligo and protein (Fig-
ure 1(a)) with highest intensity of signal observed at the
highest concentration (10 nM) of protein and oligo used.
Further, 384-well microplate was employed to down size
the volume and increase the throughput, as well as vali-
date the results of 96-well format and determine the
hooking point. At the hook point, the association and the
dissociation of the target molecule with either donor or
acceptor beads is equal, hence a maximum signal is de-
tected (Figure 1(b)). The amount of beads (~1015) used
is significantly less than the amount of protein/DNA used
and also in the right ratio so that all the protein and DNA
are bound to the beads and no free protein/DNA is pre-
sent in solution. This happens only when the protein/
DNA is below 10 nM concentrations and at any concen-
tration above this, we observed significant decrease in
signal because the free protein/DNA that remains in so-
lution binds to their respective bead bound partners,
causing loss of signal (acceptor and donor beads are not
brought together) and hence causing the hook effect. The
saturation curve showed a gradual decline in signal
counts after 10 nM of concentration. Hence, the binding
for NF-κB p50 protein and β-IFN-κB binding sequence
oligo occurs below 10 nM. The “hook effect” is common
in ELISA assay that refers to measured levels of antigen
displaying a significantly lower absorbance than the ac-
tual level present in a sample [20]. To determine the
binding equilibrium for NF-κB p50 protein and β-IFN-κB
binding sequence oligo interaction, varying concentra-
tions of oligo ranging from 0.1 nM to 100 nM and 10 nm
to 60 nM of NF-κB p50 protein were titrated (Supple-
mentary Figure S4). The results of 96 wells (Figure 1(a))
and 384 wells (Figure 1(b)) plate formats were the same
(hook point at 10 nM) and the saturation curves were
homologous to the previously reported data [2,15-18]
thus, validating our methodology. Also the reproducibil-
ity of this assay was demonstrated by performing the
assay in triplicates for each of the concentrations. The
binding is measured below this hooking point and the
saturation curve can be observed as a function of binding.
Thus, we have described a methodology comprising of
protocols used in a HTP fashion which can speed up the
NF-κB protein interaction with DNA studies. It will help
in better understanding of the mechanisms of NF-kB
signaling pathwa y and the physiological functions of
activated NF-kB.
4. Acknowledgements
We thank Prof. Gorenstein (IMM) of the University of
Texas Health Science Center for helpful discussions. The
authors would like to also acknowledge support from Dr.
Kenda Evans and Dr. Dawn Mercer from Perkin Elmer,
USA for analyzing data.
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Determination of Interaction between NFκB p50 and β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion 177
Supplementary Data
Materials and Methods
Reagents
1) Anti-GST conjugated Acceptor beads (Perkin Elmer-
AL110M)
2) Streptavidin coated donor beads (Perkin Elmer-
6760002)
3) DNA construct (5’-TGGGAATTCCC-3’) (Midland
Certified Reagents, Midland, TX).
4) Purified Protein (NFκB p50) (Fujita et al., 1992)
5) MilliQ water
Instrument
1) EnSpireTM Alphaplus multiplate reader (PerkinElmer).
Miscellaneous
1) AlphaPlate-384 (PerkinElmer-6005350)
3) Pipettes Thermo Scientific (2 - 10 μl - 10 - 100 μl)
4) Pipette tips (10 μl - 200 μl)
Figure S1. Schematic representation of AlphaLISA screen of NF-KB p50 binding sequence oligo motif Biotinylated-
TGGGAATTCCC.
Figure S2. Flowchart explaining the protocol for AlphaLISA screens (96 wells format). 1. Mix 10 μl oligo and 10 ul protein. 2.
Add 15 μl of Anti-GST conjugate acceptor beads and incubate it for one hour at 23˚C to allow the conjugation between the
beads and the protein. 3. Add 15 μl streptavidin coated donor beads (20 mg/ml final concentration) and again incubate it for
one hour at 23˚C in dark since the beads are light sensitive and lastly. 4. Reading of samples in EnSpireTM Alphaplus multi-
plate reader (PerkinElmer).
Figure S3. Plate map of EnSpire Alphaplus (96 wells format): The highest peak was observed at 10 nM concentration of both
DNA and protein. The maximum reading is highlighted by violet color square box (initial reaction was performed in dupli-
cate). Data Analysis: The dynamic range for NF-κB DNA motif Biotinylated-TGGGAATTCCC assay were determined by
preparing a diluted series ranging from 0.3 nM concentration of both protein and oligo. The data was generated using En-
SpireTM Alphaplus reader (Figure S3). The binding equilibrium constant of the given protein-oligo interaction was deter-
mined by plotting a graph of the signal obtained from the plate reader against the DNA-protein concentration.
Copyright © 2013 SciRes. JASMI
Determination of Interaction between NFκB p50 and β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion
178
Figure S4. Flowchart explaining the protocol for AlphaLISA screens (384 wells format). 1. Mix 5 μl oligo and 5 μl protein. 2.
Add 7.5 μl of Anti-GST conjugate acceptor beads and incubate it for one hour at 23˚C to allow the conjugation between the
beads and the protein. 3. Add 7.5 μl streptavidin coated donor beads (20 mg/ml final concentration) and again incubate it for
one hour at 23˚C in dark since the beads are light sensitive and lastly. 4. Reading of samples in EnSpireTM Alphaplus multi-
plate reader (PerkinElmer).
Figure S5. Plate map of EnSpireTM Alphaplus (384 wells format): The highest peak was observed at 10 nM (red circles high-
lighted by blue coloured square box) concentration of both DNA and protein and gradually started declining (orange to yel-
low to light green) with the increase in concentration of both DNA and protein. The assay was demonstrated by consistent
readings obtained for each combination performed in triplets. Data Analysis: In 384 wells plate of analytes used was half the
amount used in 96 wells plate. Also the range of concentration was broadened from 0.1 nM to 100 nM. The data was gener-
ated using EnSpire Alphaplus reader (Figure S5). The binding equilibrium constant and the “hooking point” of the given
protein-oligo interaction was determined by plotting graph of the signal obtained from the plate reader against the
DNA-protein concentration. The saturation curve showed a gradual decline in signal counts after 10 nM of concentration.
The gradual decline is due to saturation of beads with analyte. Excess analyte disrupts association between Donor and Ac-
ceptor beads beyond hook point. Thus the binding equilibrium constant for NF-κB p50 protein and B-IFN-κB oligo is 10 nM.
Copyright © 2013 SciRes. JASMI