AlphaLISA Tau-Ser400 O-GlcNAc hydrolase (OGA) Assay

T E C H N I C A L
N O T E
AlphaLISA #28
AlphaLISA Tau-Ser400 O-GlcNAc
hydrolase (OGA) Assay
Authors
Marie-Élaine Caruso
Mireille Caron
Nancy Gauthier
Anja Rodenbrock
Philippe Bourgeois
Liliana Pedro
Lucille Beaudet
Roberto Rodriguez-Suarez
AlphaLISA® Technology
PerkinElmer, Inc.
Montreal, QC
Canada, H3J 1R4
This AlphaLISA immunodetection assay measures the hydrolysis of the
O-GlcNAc moiety from a biotinylated Tau-Ser400-O-GlcNAc peptide.
Anti-O-linked-GlcNAc AlphaLISA Acceptor Beads
• AL144C: 250 µg; 500 assay points*
• AL144M: 5 mg, 10,000 assay points*
• AL144R: 25 mg, 50,000 assay points*
*0.5 µg/assay point
Peptidic Substrate Sequence
KWKHGAEIVYKSPVV-S(O-GlcNAc)-GDTSPRHLSNVK-K(biotin)-NH2
GlcNac
S/T
B
No
Enzyme
+ O-GlcNAc
Hydrolase
GlcNac
S/T
B
B
+ SA-Donor Bead
+ Ab-Acceptor Bead
S/T
+ SA-Donor Bead
+ Ab-Acceptor Bead
Emission
615 nm
Excitation
680 nm
Excitation
680 nm
No
Emission
GlcNac
B
S/T
B
S/T
Figure 1. Schematic representation of the AlphaLISA detection of an O-GlcNAcylated peptide
(B: biotin group; S/T: serine or threonine residue).
AlphaLISA Assays
AlphaLISA technology is a powerful and versatile
platform that offers highly sensitive, no-wash
immunoassays using Alpha Donor and AlphaLISA
Acceptor beads. In this technical note, we present
the optimization of an OGA signal-decrease assay
using as substrate a biotinylated Tau-derived peptide
O-GlcNAcylated at Ser400. In this assay, detection
of the non-hydrolyzed O-GlcNAcylated substrate was
performed by the addition of Streptavidin (SA) Alpha
Donor beads and AlphaLISA Acceptor beads conjugated
to an antibody (Ab) directed against the O-linkedGlcNAc moiety. Upon laser irradiation of the beadstarget complexes at 680 nm, short-lived singlet oxygen
molecules produced by the Donor beads can reach the
Acceptor beads in proximity to generate an amplified
chemiluminescent signal at 615 nm. The signal decrease
is proportional to the activity of the OGA enzyme.
Development of an AlphaLISA Tau-Ser400 O-GlcNAc
hydrolase (OGA) Assay
Reagents needed for the assay:
AlphaLISA anti-O-linked-GlcNAc Acceptor beads
Alpha Streptavidin Donor beads
Tau-Ser400-O-GlcNAc (388-411), biotinylated
AlphaLISA 5X Epigenetics Buffer 1 Kit
O-GlcNAcase (S. pyogenes), recombinant (OGA)
White opaque OptiPlate™-384
TopSeal™-A film
O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)
amino N-phenyl carbamate (PUGNAc)
N6-Methyladenine
Experiment 1: Enzyme Titration and Time-Course
PerkinElmer # AL144
PerkinElmer # 6760002
AnaSpec # 65409
PerkinElmer # AL008
Prozomix # PRO-E0255
PerkinElmer # 6007290
PerkinElmer # 6050195
Carbosynth EA06838
Carbosynth FM10151
Assay Buffer: 20 mM MES pH 6.5, 0.05% BSA and 0.01% Tween
Standard Protocol
• Dilute OGA enzyme, inhibitors and biotinylated Tau-Ser400-O-GlcNAc
peptide substrate in Assay Buffer just before use.
• Add to the wells of a white OptiPlate-384:
– 2.5 μL of inhibitor (4X) or Assay Buffer
– 2.5 μL of enzyme (4X)
– Incubate for 10 min at 23 °C.
– 5 μL of biotinylated Tau-Ser400-O-GlcNAc peptide (2X)
• Cover the plate with TopSeal-A film and incubate at 37 °C.
• Prepare 1X Epigenetics Buffer 1 as recommended in the buffer technical
data sheet.
• Prepare Acceptor beads at 100 µg/mL in 1X Epigenetics Buffer 1 (final
concentration of 20 µg/mL in 25 µL total assay volume).
• Add 5 μL of Acceptor beads. Addition of Acceptor beads prepared in
Epigenetics Buffer 1 stops the enzymatic reaction.
• Cover with TopSeal-A film and incubate 60 min at 23 °C.
• Prepare Streptavidin Donor beads at 50 µg/mL in 1X Epigenetics
Buffer 1 in subdued light (final concentration of 20 µg/mL in 25 µL
total assay volume).
• Add 10 µL of Donor beads in subdued light.
• Cover with TopSeal-A film and incubate in subdued light for 30 min at 23 °C.
• Read signal in Alpha mode with the EnVision® or EnSpire® readers.
Enzymatic progress curves were performed by incubating OGA at concentrations
ranging from 0.1 to 3 nM with 50 nM biotinylated Tau-Ser400-O-GlcNAc
peptide substrate. Enzymatic reactions contain 1% DMSO. Acceptor beads were
added at the indicated times. Donor beads were added 60 min later and signal
was read after 30 min. A 30 min reaction time using 1 nM enzyme was selected
for all subsequent experiments.
Experiment 2: Enzyme Inhibition
Serial dilutions of PUGNAc and N6-Methyladenine ranging from 300 pM to
10 µM and 10 nM to 1 mM, respectively, were pre-incubated for 10 min with
1 nM nM OGA. Enzymatic reactions were initiated by the addition of 50 nM
biotinylated Tau-Ser400-O-GlcNAc peptide substrate. Enzymatic reactions
contain 1% DMSO.
Experiment 3: Z'-factor Determination
OGA (1 nM) was pre-incubated with or without 3 µM PUGNAc or 300 µM
N6-Methyladenine for 10 min. Enzymatic reactions were initiated by the
addition of 50 nM biotinylated Tau-Ser400-O-GlcNAc peptide substrate.
Enzymatic reactions contain 1% DMSO.
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