Determination of Phospholipase Activity using Scintillation Proximity Assay

A Homogeneous TR-FRET NF-κB Protein:DNA
Binding Assay
Introduction
Figure 1. NF-κB binding assay schematic
Excitation at 340nm
Eu3+
340/35nm excitation and 670/11nm emission filter sets.
Lag time was set at 50µS, integration time was 400µS
with 30 flashes/well.
DMSO is a solvent commonly used in assay buffers and
preparations. Exposing the sample wells in an assay to
varying amounts of the solvent was performed to evaluate
of the tolerance of the assay to DMSO. DMSO was added
to the sample wells at a maximal concentration of 10% of
the well volume.
TR-FRET
P65 GST
40000
0.0
Lowest limit of detection (sensitivity) of NF-κB specific
dsDNA in the assay was 5.2fmol/well (52pM) as
determined by titration with 10nM recombinant protein
(figure 2). At maximal acceptor concentration (40nM),
signal:noise for the assay was 9.6:1.
5.0
6.0
7.0
8.0
9.0 10.0
Z’ factor analysis was performed as described by
Zhang et al4. Results of the analysis are shown in
figure 6. Assays with a Z’ factor between 0.5 and 1
are considered to be robust and reliable. The NF-κB
protein:DNA binding assay that we have developed
has a Z’ factor of 0.87.
Figure 5. IκBα protein inhibition.
1 0 0
8 0
30000
7 0
3
L o g
20000
10000
0
0
1
2
3
1 0
4
Iκ B
5
c o n c e n t r a t io n
(p M )
IκBα protein was titrated with a constant 10nM p65-GST recombinant
protein at a maximum concentration of 100nM. IC50 value determined from
the assay was 15nM. Data was plotted as triplicates, mean ± SEM.
S e n si t i v i t y = 5 . 2 f m o l / w e l l
4
Figure 6. Z’ Analysis.
p m o l/w e ll N F κ B s p e c if ic
dsDNA
60000
Specific biotinylated dsDNA was titrated from 40nM with a constant 10nM p65GST and europium chelate labelled anti-GST antibody. Cy5 labelled streptavidin
acceptor was added to the biotinylated dsDNA at a 1:2 (w/w) concentration. Data
plotted as quadruplicates, mean ± SEM.
Figure 3. NF-κB binding competition.
TR-FRET RFU
TR-FRET RFU
40000
Oligonucleotide sequences were prepared ‘in house’
using standard phosphoramidite chemistry. The 19
base-pair stretch of double-stranded DNA’s yielded are
shown in Table 1.
40000
20000
100
0
0
80
20
40
60
80
100
120
140
%B/B0
R e p l ic a t e
60
10nM of p65-GST recombinant protein was incubated with 10nM of Eu
anti-GST antibody and 10nM Cy5 streptavidin in the presence („) or
absence (À) of NF-κB specific dsDNA. Z’ factor analysis was evaluated
over 140 sample wells. Z’ = 0.87.
40
20
0
Evaluation of the assay to DMSO tolerance showed that
no significant effect was evident even at the highest
DMSO content of 10% of the volume in the well (figure 4).
CONCLUSION
We have demonstrated the ability to measure
•
NF-κB specific dsDNA binding to a p65-GST
recombinant protein by TR-FRET using generic
reagents and measuring on FARCyte.
The assay is both robust and sensitive using
•
generic TR-FRET reagents.
The results highlight the potential of the
•
FARCyte Fluorescence Plate Reader for
measurement of sensitive TR-FRET assays.
Inhibition of protein:dsDNA binding was evaluated using
IκBα protein. IκBα is a NFκB regulatory protein found in
the cell cytoplasm which inhibits its DNA binding activity.
Effect of inhibition by IκBα is shown in figure 5 and
demonstrates that increasing concentrations of the
inhibitory protein reduces the amount of signal in the
assay as less binding occurs. IC50 values for the inhibition
of NFκB specific dsDNA binding to the p65 recombinant
protein by IκBα in the assay was 15nM.
References
1.
Bours, V., et al., Biochemical Pharm., 60,
1085-1090, (2000).
2.
Baldwin, A. S., J. Clin. Invest. 107, 1, 3-6,
(2001).
3.
Jones, S. G., et al., J. Fluoresc. 11,1,13-21,
(2001)
4.
Zhang, J-H., et al., J Biomol Screening, 2, 6773 (1999).
2
NF-κB p65-GST recombinant protein (10nM) was
incubated with Eu anti-GST fusion protein (10nM) in the
dark with agitation. The reaction mixture was incubated
for 1 hour at room temperature (20-25oC) in 10mM
HEPES, 20mM Sodium Acetate, 0.2mM EDTA buffer,
pH 7.0 containing 5mM DTT, 1mg/ml BSA and 0.05%
NP40.
Sensitivity was evaluated using a 2n titration of 40nM of
biotinylated NF-κB specific dsDNA. For competition,
inhibition, DMSO tolerance and Z’ analysis assays,
20nM biotinylated NF-κB specific dsDNA was added to
the reaction mix and incubated at room temperature for
a further 1 hour in the dark with agitation. Finally, Cy5
streptavidin (10nM) was added to each reaction well
and further incubated for 15 minutes. Reactions were
performed in a total volume of 100µl using Corning
black 384-well NBS plates. TR-FRET was measured on
the FARCyte fluorescence plate reader using
4.0
Evaluation of the effect of DMSO solvent on the assay signal. Data is
plotted as quadruplicates ± SEM.
Cy5 labelled streptavidin
Oligo sequences were prepared using standard phosphoramidite chemistry
and purified by C18 reverse phase HPLC. Double-stranded DNA was
prepared by incubating equimolar amounts of the NF-κB-specific (HIV-L)
coding (biotinylated or non-biotinylated) and unmodified non-coding strands in
o
a 75 C water bath for 3-5 minutes before allowing to cool to ambient
temperature.
3.0
%B/B0
Biotin dsDNA
Unlabelled NF-κB-specific competitor dsDNA
5’
GATCTAGGGACTTTCCGCG 3’
3’
ATCCCTGAAAGGCGCCTAG 5’
Unlabelled non-specific competitor dsDNA
5’
GATCTATTGACTTAAGTG 3’
3’
ATAACTGAATTCACCTAG 5’
2.0
9 0
50000
Table 1. NF-κB assay dsDNA
Biotinylated dsDNA (NF-κB-specific)
5’ Biotin-GATCTAGGGACTTTCCGCG 3’
3’
AT CCCTGAAAGGCGCCTAG 5’
1.0
% DM SO
Figure 2. NF-κB assay sensitivity of binding.
Method
20000
0
Emission at 665nm
Assay is configured using a europium chelate labelled anti-GST antibody
specific for a p65-GST recombinant protein that interacts with a biotinylated
NFκB-specific dsDNA bound to Cy5 labelled streptavidin
30000
10000
Results
Competition of binding to the p65-GST protein (figure 3)
demonstrated that signal is significantly reduced with the
addition of the specific dsDNA competitor containing the
NF-κB p65 consensus binding sequence, whereas the
non-specific dsDNA reduced signal only marginally when
added at considerable excess, demonstrating binding
specificity.
TMT(Eu) Labelled Anti-GST
Figure 4. Effect of DMSO on the assay.
TR-FRET RFU
Nuclear Factor-κB (NF-κB) is a transcription factor that
is considered to be of physiological importance
because of its key role as a regulatory molecule
involved in immune response, inflammation, cancer and
apoptosis1,2.
We have developed a time resolved-fluorescence
resonance energy transfer (TR-FRET) assay to
evaluate the binding interaction between the p65
subunit of NF-κB and a dsDNA NFκB-specific (HIV-L)
consensus sequence.
The development of a TR-FRET NF-κB binding assay
has been reported previously3 using direct labelled p65
specific dsDNA. We have now further developed the
assay to incorporate generic europium [Eu(TMT)]
donor and Cy5 acceptor reagents (figure 1).
3
Lo g
10
4
5
6
7
d s D N A c o n c e n tr a t io n (p M )
Competition of the biotinylated dsDNA for binding to the p65-GST recombinant
protein was demonstrated using unlabelled NF-κB specific dsDNA sequence („)
and an unlabelled non-specific dsDNA sequence (À). dsDNA was titrated from a
maximum concentration of 1µM. Data plotted as triplicates, mean ± SEM.
Copyright ©2009, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc.
All other trademarks are the property of their respective owners.
008908_10
Similar pages