LANCE Ultra PRMT4 (CARM1) Histone H3-Arginine N ... Lance_52_final PRMT4.pdfTECHNICAL NOTE LANCE...

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TECHNICAL NOTE LANCE Ultra PRMT4 (CARM1) Histone H3-Arginine N-methyltransferase Assay This LANCE Ultra immunodetection assay measures the methylation of a biotinylated histone H3 (21-44) peptide at arginine 26. Europium-anti-methyl-Arginine Antibody • TRF0415-D: 10 μg, 1,562 assay points* • TRF0415-M: 100 µg, 15,620 assay points* *40 fmol/assay point Peptidic Substrate Sequence: ATKAARKSAPSTGGVKKPHRYRPG-GK(Biotin)-NH2 LANCE Ultra Assays LANCE Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assays use a proprietary europium chelate donor dye, W1024 (Eu), together with ULight , a small molecular weight acceptor dye with a red-shifted fluorescent emission. In this technical note, we present the optimization of a PRMT4 assay using a biotinylated histone H3-derived peptide as substrate. Detection of the histone H3 arginine 26 methylated product was performed by the addition of the Eu-labeled antibody (Eu-Ab) and ULight-Streptavidin (ULight-SA), which bring the Eu donor and ULight acceptor dye molecules into close proximity. Upon irradiation at 320 or 340 nm, the energy from the Eu donor is transferred to the ULight acceptor dye which, in turn, generates light at 665 nm. The signal intensity is proportional to the activity of the PRMT4 enzyme. LANCE ® Ultra TR-FRET Technology U-TRF #52 + Enzyme SAM SAH B R Me B R Me B R ULight Emission 665 nm Excitation 320 or 340 nm Eu + Eu-Ab + ULight-SA Figure 1. Schematic representation of the LANCE Ultra detection of a methylated histone peptide (B: biotin group; R: arginine residue). Nancy Gauthier Jean-Philippe Lévesque-Sergerie Philippe Bourgeois Jean-François Michaud Liliana Pedro Marie-Élaine Caruso Anja Rodenbrock Lucille Beaudet Roberto Rodriguez-Suarez PerkinElmer, Inc. Montreal, QC Canada H3J 1R4

Transcript of LANCE Ultra PRMT4 (CARM1) Histone H3-Arginine N ... Lance_52_final PRMT4.pdfTECHNICAL NOTE LANCE...

Page 1: LANCE Ultra PRMT4 (CARM1) Histone H3-Arginine N ... Lance_52_final PRMT4.pdfTECHNICAL NOTE LANCE Ultra PRMT4 (CARM1) Histone H3-Arginine N-methyltransferase Assay This LANCE Ultra

T E C H N I C A L N O T E

LANCE Ultra PRMT4 (CARM1) Histone H3-Arginine N-methyltransferase Assay

This LANCE Ultra immunodetection assay measures the methylation of a biotinylated histone H3 (21-44) peptide at arginine 26.

Europium-anti-methyl-Arginine Antibody

• TRF0415-D:10μg,1,562assaypoints*

• TRF0415-M:100µg,15,620assaypoints*

*40 fmol/assay point

Peptidic Substrate Sequence:

ATKAARKSAPSTGGVKKPHRYRPG-GK(Biotin)-NH2

LANCE Ultra Assays

LANCE Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assays use a proprietary europium chelate donor dye, W1024 (Eu), together with ULight™, a small molecular weight acceptor dye with a red-shifted fluorescent emission. In this technical note, we present the optimization of a PRMT4 assay using a biotinylated histone H3-derived peptide as substrate. Detection of the histone H3 arginine 26 methylated product was performed by the addition of the Eu-labeled antibody (Eu-Ab) and ULight-Streptavidin (ULight-SA), which bring the Eu donor and ULight acceptor dye molecules into close proximity. Upon irradiation at 320 or 340 nm, the energy from the Eu donor is transferred to the ULight acceptor dye which, in turn, generates light at 665 nm. The signal intensity is proportional to the activity of the PRMT4 enzyme.

LANCE® Ultra TR-FRET Technology

U-TRF#52

+ EnzymeSAM

SAH

B RMe

B RMe

B R

ULightEmission665 nm

Excitation320 or 340 nm

Eu

+ Eu-Ab+ ULight-SA

Figure 1. Schematic representation of the LANCE Ultra detection of a methylated histone peptide (B: biotin group; R: arginine residue).

Nancy Gauthier Jean-Philippe Lévesque-SergeriePhilippe Bourgeois Jean-François MichaudLiliana Pedro Marie-Élaine Caruso Anja Rodenbrock Lucille Beaudet Roberto Rodriguez-Suarez

PerkinElmer, Inc.Montreal, QCCanada H3J 1R4

Page 2: LANCE Ultra PRMT4 (CARM1) Histone H3-Arginine N ... Lance_52_final PRMT4.pdfTECHNICAL NOTE LANCE Ultra PRMT4 (CARM1) Histone H3-Arginine N-methyltransferase Assay This LANCE Ultra

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Development of a PRMT4 Histone H3-Arginine N-methyltransferase Assay

Reagents needed for this assay:

Europium anti-methyl-Arginine Antibody PerkinElmer # TRF0415LANCE Ultra ULight-Streptavidin PerkinElmer # TRF0102Histone H3 (21-44) peptide, amide, biotinylated AnaSpec # 64641LANCE Detection Buffer, 10X PerkinElmer # CR97-100PRMT4 (human CARM1), recombinant Reaction Biology # HMT-11-120White opaque OptiPlate™-384 PerkinElmer # 6007290TopSeal™-A film PerkinElmer # 6050195S-adenosylmethionine (SAM) Sigma # A7007S-(5'-Adenosyl)-L-homocysteine (SAH) Sigma # A9384Sinefungin Sigma # S8559Suramin Tocris Bioscience # 1472AMI-1 Enzo Life Sciences # ALX-270-440SAM is prepared at 30 mM in 5 mM H2SO4/10% ethanol (v/v) in H2O.

Assay Buffer: 25 mM Tris-HCl, pH 8.0, 1 mM DTT, 0.01% BSA, 0.01% Tween-20

Standard Protocol• DilutePRMT4enzyme,SAM,inhibitorsandbiotinylatedhistone

H3 (21-44) peptide substrate in Assay Buffer just before use.• AddtothewellsofawhiteOptiPlate-384: –5μLofinhibitor(2X)orAssayBuffer –2.5μLofenzyme(4X) – Incubate for 10 min at room temperature (RT). –2.5μLofbiotinylatedhistoneH3(21-44)-NH2 peptide/SAM mix (4X) For SAM titration, add SAM dilutions independently of substrate.• CovertheplatewithTopSeal-AfilmandincubateatRT.• PrepareDetectionMixbydilutingtheEu-Abto4nM,ULight-Streptavidin

to 100 nM in 1X LANCE Detection Buffer (final concentration of 2 nM and 50 nM, respectively, in 20 µL total assay volume).

• Add10μLofDetectionMix.Addition of the Detection Mix stops the enzymatic reaction.

• CoverwithTopSeal-Afilmandincubate60minatRT.• RemovetheTopSeal-AfilmandreadsignalwiththeEnVision® Multilabel

Plate Reader in TR-FRET mode (excitation at 320 or 340 nm and emission at 665 nm).

Experiment 1: Enzyme Titration and Time-Course

Results

Enzymatic progress curves were performed by incubating PRMT4 at concentrations ranging from 2 to 50 nM with 30 nM biotinylated histone H3 peptide substrate plus 100 µM SAM. Reactions were stopped by the addition of the Detection Mix at the indicated times. Signal was read after 60 min. A 60 min reaction time using 20 nM enzyme was selected for all subsequent experiments.

Experiment 2: SAM Titration

Serial dilutions of SAM ranging from 10 nM to 100 µM were added to 20 nM PRMT4 and 30 nM biotinylated histone H3 peptide substrate. A 1 µM SAM concentration was selected for subsequent experiments.

Experiment 4: Z’-factor Determination

PRMT4 (20 nM) was pre-incubated with or without 100 µM SAH for 10 min. Enzymatic reactions were initiated by the addition of 30 nM biotinylated histone H3 peptide substrate plus 1 µM SAM. Enzymatic reactions contain 1% DMSO.

Experiment 3: Enzyme Inhibition

Serial dilutions of inhibitors ranging from 0.1 nM to 10 µM (suramin) and 1 nM to 100 µM (sinefungin, SAH and AMI-1) were pre-incubated for 10 min with 20 nM PRMT4. Enzymatic reactions were initiated by the addition of 30 nM biotinylated histone H3 peptide substrate plus 1 µM SAM. Enzymatic reactions contain 1% DMSO.