Determination of Interaction between NFκB p50 and <i>β</i>-IFN-<i>κ</i>B Binding Oligo Using AlphaLISA in HTP Fashion

doi:10.4236/jasmi.2013.33022

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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: *[email protected]

Received July 28th, 2013; revised August 25th, 2013; accepted September 1st, 2013

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.

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.

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.

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.