TECHNICAL NOTE AlphaLISA H3K4 (unmodified) Cell-based Assay Control Kit Introduction This AlphaLISA ® immunodetection assay monitors unmodified histone H3 lysine 4 (H3K4) in cellular extracts. It is intended to be used as control for assessing total histone H3 levels when detecting epigenetic marks on cellular histone H3 using Alpha technologies. AlphaLISA Assays The AlphaLISA technology allows performing no-wash homogeneous proximity immunoassays using Alpha Donor and AlphaLISA Acceptor beads. Following a homogeneous histone extraction protocol, the mark of interest is detected by the addition of a biotinylated anti- Histone H3 (C-terminus) antibody and AlphaLISA Acceptor beads conjugated to an antibody (Ab) specific to the mark. The biotinylated antibody is then captured by Streptavidin (SA) Donor beads, bringing the two beads into proximity. Upon laser irradiation of the Donor beads 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. In this technical note, we describe an optimized AlphaLISA unmodified H3K4 cellular detection assay used for normalizing data obtained with AlphaLISA assays targeting different histone H3 marks. In normally growing HeLa cells, the histone H3 lysine 4 modification stoichiometry is less than 8%, thus leaving more than 92% of the lysine 4 residue completely unmodified. (Peach et al. Mol Cell Proteomics. 2012 11:128-37). Hence, this mark becomes highly suitable for evaluating total histone H3 levels. We present here the monitoring by AlphaLISA AlphaLISA ® Technology AlphaLISA #24 Authors Nathalie Rouleau Philippe Bourgeois Nancy MacDonald Jean-Francois Michaud Jean-Philippe Levesque-Sergerie Lucille Beaudet Mathieu Arcand PerkinElmer, Inc. Montreal, QC Canada, H3J 1R4 Excitation 680 nm Emission 615 nm B K Anti-H3K4 Acceptor Streptavidin Donor Extracted Histone H3 Biotin Anti-H3 Figure 1. Schematic representation of the AlphaLISA cellular assay for the detection of histone proteins. of unmodified H3K4 levels in seven cell lines with corroborating data from Western blot analysis. These results confirmed that levels of both unmodified H3K4 and total histone H3 are present in equivalent amounts in all seven cell lines. We then assessed by AlphaLISA and Western blot analysis changes in the levels of H3K9me2 and H3K79me2 following treatment of cells with 3-Deazaneplanocin A (DZNep) and UNC0638, two histone methyltransferase (HMT) inhibitors, and with the histone demethylase inhibitors IOX1 and deferoxamine (DFX). Both methods demonstrated that while H3K9me2 and H3K79me2 mark modulation is induced by these four compounds, unmodified H3K4 levels remain unaffected. Taken together, our data indicate that the AlphaLISA unmodified H3K4 cellular detection kit is an ideal tool for monitoring levels of total histone H3 present in cells.
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T E C H N I C A L N O T E
AlphaLISA H3K4 (unmodified) Cell-based Assay Control Kit
IntroductionThis AlphaLISA® immunodetection assay monitors unmodified histone H3 lysine 4 (H3K4) in cellular extracts. It is intended to be used as control for assessing total histone H3 levels when detecting epigenetic marks on cellular histone H3 using Alpha technologies.
AlphaLISA AssaysThe AlphaLISA technology allows performing no-wash homogeneous proximity immunoassays using Alpha Donor and AlphaLISA Acceptor beads. Following a homogeneous histone extraction protocol, the mark of interest is detected by the addition of a biotinylated anti-Histone H3 (C-terminus) antibody and AlphaLISA Acceptor beads conjugated to an antibody (Ab) specific to the mark. The biotinylated antibody is then captured by Streptavidin (SA) Donor beads, bringing the two beads into proximity. Upon laser irradiation of the Donor beads 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.
In this technical note, we describe an optimized AlphaLISA unmodified H3K4 cellular detection assay used for normalizing data obtained with AlphaLISA assays targeting different histone H3 marks. In normally growing HeLa cells, the histone H3 lysine 4 modification stoichiometry is less than 8%, thus leaving more than 92% of the lysine 4 residue completely unmodified. (Peach et al. Mol Cell Proteomics. 2012 11:128-37). Hence, this mark becomes highly suitable for evaluating total histone H3 levels. We present here the monitoring by AlphaLISA
AlphaLISA® Technology
AlphaLISA #24
Authors
Nathalie RouleauPhilippe BourgeoisNancy MacDonaldJean-Francois MichaudJean-Philippe Levesque-Sergerie Lucille Beaudet Mathieu Arcand PerkinElmer, Inc.Montreal, QC Canada, H3J 1R4
Excitation680 nm
Emission615 nm
B
K
Anti-H3K4 Acceptor
Streptavidin Donor
Extracted Histone H3
BiotinAnti-H3
Figure 1. Schematic representation of the AlphaLISA cellular assay for the detection of histone proteins.
of unmodified H3K4 levels in seven cell lines with corroborating data from Western blot analysis. These results confirmed that levels of both unmodified H3K4 and total histone H3 are present in equivalent amounts in all seven cell lines. We then assessed by AlphaLISA and Western blot analysis changes in the levels of H3K9me2 and H3K79me2 following treatment of cells with 3-Deazaneplanocin A (DZNep) and UNC0638, two histone methyltransferase (HMT) inhibitors, and with the histone demethylase inhibitors IOX1 and deferoxamine (DFX). Both methods demonstrated that while H3K9me2 and H3K79me2 mark modulation is induced by these four compounds, unmodified H3K4 levels remain unaffected. Taken together, our data indicate that the AlphaLISA unmodified H3K4 cellular detection kit is an ideal tool for monitoring levels of total histone H3 present in cells.
Standard Protocol
• Distribute10µLofcellsinthewellsofaCulturPlate-384microtiterplate.
• FillouterandunfilledwellswithsterilewaterorPBS.
• Incubateadherentcellsfor3-4hat37°Cina5%CO2 atmosphere to allow cell adhesion. Skip this step for cells growing in suspension.
• Add5µLofculturemediumormodulatorpreparedat3Xitsfinalconcentration in medium.
• Incubatecellsfor16-72hat37°Cina5%CO2 atmosphere. For longer incubation, a medium change is recommended.
• Add5µLofCell-Histone™ Lysis buffer.
• Incubate15minatroomtemperature.
• Add10µLofCell-HistoneExtractionbuffer.
• Incubate10minatroomtemperature.
• Dilutethe10XCell-HistoneDetectionbufferto1Xwithwater.
• Preparea5XmixofAcceptorbeadsat100µg/mLandbiotinylatedanti-Histone H3 at 15 nM in 1X Cell-Histone Detection buffer.
• Add10µLofthe5XmixofAcceptorbeads/biotinanti-HistoneH3antibody(finalconcentration20µg/mLand3nM,respectively).
• CoverwithTopSeal-Afilmandincubatefor60minat23°C.
• Prepareinsubduedlighta5XsolutionofSADonorbeadsat100µg/mLin 1X Cell-Histone Detection buffer.
• Add10µLDonorbeads(finalconcentration20µg/mL).
• CoverwithTopSeal-Afilmandincubatefor30minat23°Cinthedark.
• ReadsignalinAlphamodewiththeEnVision®orEnSpire® Multilabel platereader.Platescanbereadafterovernight(O/N)incubationtomaximize signal intensity.
Detection of Histone H3 in Cellular ExtractsReagents needed for the assay:
AlphaLISA Unmodified Histone H3 Lysine4(H3K4)CellularDetectionKit PerkinElmer#AL719
AlphaLISA Di-Methyl-Histone H3 Lysine9(H3K9me2)CellularDetectionKit PerkinElmer#AL717
AlphaLISA Di-Methyl-Histone H3 Lysine 79 (H3K79me2)CellularDetectionKit PerkinElmer#AL748
White opaque CulturPlate™-384 PerkinElmer#6007680
TopSeal™-Afilm PerkinElmer#6050195
Deferoxaminemesylatesalt(DFX) Sigma#D9533
3-DeazaneplanocinA(DZNep) CaymanChemical#13828
UNC0638hydrate Sigma#U4885
5-Carboxy-8-hydroxyquinoline(IOX1) Sigma#SML0067
PolyScreen®PVDFHybridizationTransfer Membraneformini-gels PerkinElmer#NEF1003001PK
Western Lightning™ECLPro PerkinElmer#NEL120001EA
Anti-MouseIgG(Goat),HRP-Labeled PerkinElmer#NEF822001EA
Anti-RabbitIgG(Goat),HRP-Labeled PerkinElmer#NEF812001EA
Cell Lines:
HeLa ATCC#CCL-2.2™
KYSE-150 DSMZ#ACC375
MCF7 ATCC#HTB-22
U-2OS ATCC#HTB-96
MDA-MB-231 ATCC#HTB-26
OCI-LY-19 DSMZ#ACC528
SU-DHL-6 DSMZ#ACC572
Cell seeding medium:
HeLaandMCF7:MEM/EBSS HyClone#SH30024 supplemented with 10% FBS.
KYSE-150HAM’sF12 HyClone#SH30026 supplemented with 10% FBS
OCI-LY-19andSU-DHL-6:AlphaMEM HyClone#SH30265 supplemented with 15% FBS
2
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Experiment 2: Specificity of Cellular Detection
A) AlphaLISA assays. HeLa cells were seeded at a density of 1,000 cells per well and treated for 48 h with the JMJD2 inhibitors IOX1 or DFX (from 1 nM to 100 µM), the broadly-selective HMT inhibitor DZNep (from 300 pM to 30 µM) or the G9a inhibitor UNC0638 (from 100 pM to 3 µM) in medium containing 0.3% DMSO. A) Detection of the H3K9me2 (top) and H3K79me2 (middle) marks. Detection of unmodified H3K4 (bottom), which represents over 90% of all H3K4 marks, was used to assess total histone H3 levels. B) Western blot analysis of H3K4, H3K9me2, H3K79me2 and total histone H3 levels in HeLa cells treated with DZNep (30 µM), UNC0638 (1 µM), IOX1 (30 µM) or 0.3% DMSO (untreated controls). Although increase (IOX1 and DFX) and decrease (DZNep and UNC0638) of the H3K9me2 and H3K79me2 marks were observed in both AlphaLISA and Western blot, there was no significant change in unmodified H3K4 mark levels, concomitant with total histone H3 levels assessed by Western blot.
Experiment 3: Compound-Induced Modulation
HeLa cells were seeded at a density of 2,000 cells/well. Serial dilutions of histone H3-derived peptides bearing various epigenetic marks were added to the wells at concentrations ranging from 100 pM to 10 μM immediately before the addition of the AlphaLISA detection reagents. With the exception of H3pT3, all peptides unmodified at lysine 4 competed for the interaction between the Acceptor beads and histone H3 proteins with IC50 values ranging from 0.3 to 0.6 µM.
Experiment 1: Detection of Histone H3
Evaluation of total histone H3 levels in HeLa, KYSE-150, MCF7 U-2 OS, MDA-MB-231, OCI-LY-19 and SU-DHL-6 cells. A) AlphaLISA detection of unmodified H3K4 in serial dilutions of cell extracts corresponding to densities ranging from 500 to 20,000 cells per well. Cell-Histone Lysis as well as Extraction buffers were sequentially added prior to detection reagents in accordance with the standard protocol. B) Western blot detection of unmodified H3K4 and total histone H3 levels. Cell lysates were separated by SDS-PAGE on a 10%-20% gradient gel. Following transfer to PolyScreen membrane, histone H3 proteins unmodified at lysine 4 were detected using the same antibody present on the Acceptor beads. For total histone H3, an antibody recognizing a histone H3 C-terminal epitope was used. Western blots were revealed using HRP-labeled-labeled anti-species secondary antibody and Western Lightning ECL Pro substrate.
Results
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