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LANCE Epigenetic Assays

Overview


LANCE® Ultra epigenetics toolbox reagents were designed for the detection of specific methylation and acetylation marks on histone H3 peptide substrates in a TR-FRET assay. Reagents that detect other post-translational modifications (such as acetylation of p53) are also available. In LANCE Ultra assays, a biotinylated histone peptide is used as substrate in a biochemical enzymatic reaction employing either histone methyltransferase (HMT) or acetyltransferase (HAT) enzymes. When the enzymatic reaction is stopped, the level of methylation or acetylation is determined using a Europium-labeled antibody and ULight™-streptavidin. The ULight-streptavidin captures the biotin moiety on the peptide substrate, whereas the Europium-labeled antibody recognizes the specific epigenetic mark (acetylation or methylation). Upon excitation of the Europium chelate fluorophore at 320 to 340 nm, energy can be transferred to the ULight acceptor fluorophore in proximity, resulting in the emission of light at 665 nm. The intensity of light emission is proportional to the level of substrate modification.

lance_epi_principle_ASK.jpg
Figure 1. Assay principle for LANCE Ultra epigenetics toolbox reagents. On the left, schematic representation of a LANCE Ultra lysine acetyl-transferase (HAT) assay. On the right, schematic representation of a LANCE Ultra lysine methyl-transferase (HMT) assay. In both cases, a biotinylated histone H3 peptide substrate is used in conjunction with ULight-streptavidin and a Europium-labeled antibody specific to the particular modification (acetylated or methylated residue).

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Specificity


HATs and HMTs modify their target substrates at specific residues. Acetylation typically occurs on lysine residues while methylation can occur on lysine or arginine residues. Lysine residues can be mono-, di-, or tri-methylated, while arginines can be mono-, or symmetrically or asymmetrically di-methylated.

Currently, LANCE Europium antibodies are offered for these epigenetic and other post-translationally modified targets:

Detected modificationDescription of modificationCatalog number
H3K9acHistone H3 acetylated lysine 9TRF0400
H3K27acHistone H3, acetylated on lysine 27TRF0405
H3K4Histone H3, unmodified on lysine 4TRF0404
H3K4me1-2Histone H3, mono- or di-methylated on lysine 4TRF0402
H3K9me2Histone H3, di-methylated on lysine 9TRF0403
H3K27me2-1Histone H3, mono- or di-methylated on lysine 27TRF0406
H3K27me3Histone H3, tri-methylated on lysine 27TRF0407
H3K36me2Histone H3, di-methylated on lysine 36TRF0408
p53K382acp53, acetylated on lysine 382TRF0409
H3K9/K27 Histone H3, unmodified on lysine 9 and lysine 27 TRF0411
H4Kac panAcetylated human Histone H4TRF0412
O-linked GlcNAcO-linked n-acetylglucosamineTRF0413
H4R3meHistone H4 methylated at arginine 3TRF0414
H3/H4Rme panHistone H3 and H4 methylated arginine residuesTRF0415

Specificity of Europium-labeled antibodies has been assessed in a detection assay using biotinylated peptides with different epigenetic marks. Additional information can be obtained by contacting technical support (contact information is indicated in the upper right-hand corner of this page).

h3k9ac_ASK.jpg
Figure 2. Specificity of TRF0400, which detects H3K9ac (Histone H3 peptide acetylated on lysine 9). Histone H3-derived peptides with different epigenetic marks were titrated. Signal was detected with an EnVision 2103 set in TR-FRET reading mode. The hook effect observed at higher peptide concentrations is typical of multi-component assays.  In this particular application, a hooking type of curve is generated when peptide concentrations exceed the binding capacity of the ULight-streptavidin and/or Europium-labeled antibody.

h3k4me_ASK.jpg
Figure 3. Specificity of TRF0402, which detects mono- or di-methylated states of lysine 4 on histone H3. Histone H3-derived peptides containing different epigenetic marks were titrated. Signal was detected with an EnVision 2103 set in TR-FRET reading mode.

h3k9me_ASK.jpg
Figure 4. Specificity of TRF0403, which detects H3K9me2 (Histone H3 peptide di-methylated on lysine 9). Histone H3-derived peptides with different epigenetic marks were titrated. Signal was detected with an EnVision 2103 set in TR-FRET reading mode. The hook effect observed at higher peptide concentrations is typical of multi-component assays.  Here, it occurs when peptide concentrations exceed the binding capacity of the ULight-streptavidin and/or Europium-labeled antibody.

Custom-labeled Europium antibodies may also be available upon request, and custom assay development services are offered through our OnPoint custom teams (contact information is at the bottom of this page). It is also possible to use our LANCE Europium labeling reagents and other LANCE toolbox reagents to develop your own assay.

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What do I need to run this assay?


Reagents available from PerkinElmer:

  • LANCE Ultra Europium-labeled antibody, specific to an acetylation or methylation site (refer to table in above section)
  • LANCE ULight-streptavidin (Cat. No. TRF0102)
  • 10X LANCE Detection Buffer (Cat. No. CR97-100)
  • TopSeal-A™ adhesive plate seal (Cat. No. 6050185
  • Microplates (We recommend white OptiPlate™ microplates, Cat. No. 6007299.)
  • TRF-capable plate reader (We recommend our EnVision® Multilabel Plate Reader, our VICTOR™ plate reader, or our ViewLux™ high-throughput CCD detector.)


Reagents available from AnaSpec and other suppliers:

  • Biotinylated Histone H3 or p53 peptide substrates.

Depending on the enzyme under study, you may need to use a non-modified substrate, or you may need to start with a peptide substrate bearing a specific modification (for example a peptide containing a specific mono-methylated residue). In general, we recommend using a non-modified peptide if you are performing acetylation or methylation assays, while a pre-modified substrate will be required for deacetylation and demethylation assays (for example, tri-methylated lysine 9, acetylated lysine 9, etc). Of high interest is the recent availability of anti-non-modified residue antibodies, like the Eu-labeled anti-H3K4-non-modified antibody (TRF0404). The latter allows the development of assays for HDACs acting on H3 lysine 4 , or HDMs able to fully demethylate this residue, in a very convenient signal increase mode. Before, these types of assays could only be performed in a signal decrease setup. In any case, make sure your assay is designed so that the Europium-labeled antibody could differentiate between substrate and product.

  • Additionally, you may wish to purchase a second peptide to be used as a positive detection control for your assay. For example, if you are measuring acetylation of lysine 9 on a Histone H3 peptide, you may wish to test the biotinylated acetyl-Lys9 Histone H3 peptide.

Product

Sequence

AnaSpec catalog number

Biotinylated Histone H3 peptide (residues 1-21), non-modified (bare)

ARTKQTARKSTGGKAPRKQLA-GGK(biotin)-Nh3

#61702

Biotinylated Histone H3 peptide (residues 21-44), non-modified (bare)

ATKAARKSAPATGGVKKPHRYRPG-GK(biotin)

#64440

Biotinylated Histone H3 peptide, acetylated on Lysine 9

ARTKQTAR-K(Ac)-STGGKAPRKQLA-GGK(biotin)-Nh3

#64361

Biotinylated Histone H3 peptide, acetylated on Lysine 14

ARTKQTARKSTGG-K(Ac)-APRKQLA-GGK(biotin)-Nh3

#64362

Biotinylated Histone H3 peptide, acetylated on Lysine 9 and Lysine 14

ARTKQTAR-K(Ac)-STGG-K(Ac)-APRKQLA-GGK-biotin-Nh3

#64363

Biotinylated Histone H3 peptide, acetylated on Lysine 27

ATKAAR-K(Ac)-SAPATGGVKKPHRYRPGG-K(biotin)

#64846

Biotinylated Histone H3 peptide, mono-methylated on Lysine 4

ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK-biotin-Nh3

#64355

Biotinylated Histone H3 peptide, di-methylated on Lysine 4

ART-K(Me2)-QTARKSTGGKAPRKQLA-GGK(biotin)-Nh3

#64356

Biotinylated Histone H3 peptide, tri-methylated on Lysine 4

ART-K(Me3)-QTARKSTGGKAPRKQLA-GGK(biotin)-Nh3

#64357

Biotinylated Histone H3 peptide, mono-methylated on Lysine 9

ARTKQTAR-K(Me1)-STGGKAPRKQLA-GGK(biotin)-Nh3

#64358

Biotinylated Histone H3 peptide, di-methylated on Lysine 9

ARTKQTAR-K(Me2)-STGGKAPRKQLA-GGK(biotin)-Nh3

#64359

Biotinylated Histone H3 peptide, tri-methylated on Lysine 9

ARTKQTAR-K(Me3)-STGGKAPRKQLA-GGK(biotin)-Nh3

#64360

Biotinylated Histone H3 peptide, mono-methylated on Lysine 27

ATKAAR-K(Me1)-SAPATGGVKKPHRYRPG-GK(biotin)

#64365

Biotinylated Histone H3 peptide, di-methylated on Lysine 27

ATKAAR-K(Me2)-SAPATGGVKKPHRYRPG-GK(biotin)

#64366

Biotinylated Histone H3 peptide, tri-methylated on Lysine 27

ATKAAR-K(Me3)-SAPATGGVKKPHRYRPG-GK(biotin)

#64367

Biotinylated Histone H3 peptide, di-methylated on Lysine 36

ATKAARKSAPATGGV-K(Me2)-KPHRYRPG-GK(biotin)

#64442

Biotinylated Histone H3 peptide, tri-methylated on Lysine 36

ATKAARKSAPATGGV-K(Me3)-KPHRYRPG-GK(biotin)

#64441

Biotinylated p53 peptide (residues 361-393), non-modified (bare)

Biotin-LC-GSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD-Nh3

#62530

Biotinylated p53 peptide, acetylated on Lysine 382

Biotin-KGGHLKSKKGQSTSRHK-K(Ac)-LMFKTEGPDSD-Nh3

#64869


Reagents available from various suppliers:

  • Cofactor (acetyl CoA for acetylation assays, or S-adenosylmethionine for methylation assays; refer to Tips and FAQs below to see where PerkinElmer R&D gets these reagents.)
  • Enzyme
  • Reaction buffer
  • Test compounds as desired

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Products and catalog numbers


View a listing of relevant products and catalog numbers for LANCE assays.


Protocol-in-brief


The assay is performed in two steps: first, the enzymatic assay is performed in an appropriate reaction buffer. Then, the reaction is stopped and LANCE Eu-antibody and ULight-streptavidin are added in 1X LANCE Detection buffer for the detection step.

lance_epi_workflow_ASK.jpg
Workflow for LANCE epigenetics assay

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Assay development


Assays have to be optimized using your particular enzyme/substrate pair. Examples of assay development for a number of enzymes can be found in the format of Technical Notes. The example below describes the optimization of a p300 acetyltransferase assay using the Eu-labeled anti-H3K9ac antibody (as found in the technical note).

1. Enzyme titration and time-course.

This experiment is set up using increasing concentrations of enzyme where multiple wells are filled for each enzyme concentration allowing stopping the reaction at different time points. For subsequent assay optimization, you will want to choose a time and enzyme concentration providing for a linear increase of reaction product over time.

Keep in mind that if you are performing a methylation assay, stopping the reaction at the appropriate time could be critical as several of our LANCE Europium antibodies detect specific methylation states. For example, if a methylation reaction were to proceed for a too long time period, substrate tri-methylation could occur (provided the enzyme is used at that capacity), with an eventual loss of signal if the antibody is specific for the di-methylated residue.

lance_epi_timecourse_ASK.jpg
Figure 5. Enzymatic progress curves were performed by incubating p300 at concentrations ranging from 0.025 to 1 nM with 200 nM biotinylated H3 (1-21) peptide substrate and at a non-limiting concentration of acetyl CoA (25 µM). Reactions were stopped by the addition of anacardic acid at the indicated times, followed by the Detection Mix. Signal was read after 60 min. A 30 min reaction time using 0.1 nM enzyme was selected for all subsequent experiments.

 

2. Cofactor titration.

This assay is performed to determine the apparent Km for the cofactor (either acetyl CoA for acetylation assays, or S-adenosylmethionine for methylation assays). Increasing concentrations of cofactor are tested at the optimal enzyme concentration and time point selected in the previous experiment. For most applications, you will want to select a final concentration of cofactor that is close to the apparent Km.

lance_acetyl_titration_ASK.jpg
Figure 6. Serial dilutions of acetyl CoA ranging from 10 nM to 100 µM were added to 0.1 nM p300 and 200 nM biotinylated H3 (1-21) peptide substrate. A 5 µM acetyl CoA concentration was selected for subsequent experiments.

Before moving into the testing of the effect of compounds on the studied enzyme, it is important to perform an intermediate experiment to confirm that the selected conditions (in terms of reaction time, enzyme and cofactor concentrations) still allow for a linear increase of product over time (not shown). This can be easily accomplished by performing a time-course (enzymatic) assay using the conditions selected from Figures 5 and 6.


3. Enzyme inhibition.

This assay can be used to characterize inhibitors of your enzyme, or as part of assay validation using a reference inhibitor if you will be screening. Increasing concentrations of inhibitor are tested at the enzyme concentration, time point, and cofactor concentration determined from the previous two experiments. An IC50 for the inhibitor can be derived from this data. It is important to note that if the compounds to be tested are dissolved in a solvent that is different than water (typically DMSO), its effect on the enzymatic reaction must be evaluated beforehand.

lance_epi_inhibitor_ASK.jpg
Figure 7. Serial dilutions of anacardic acid ranging from 200 nM to 100 µM and garcinol ranging from 120 nM to 120 µM were pre-incubated for 10 min with 0.1 nM p300. Enzymatic reactions were initiated by the addition of 200 nM biotinylated H3 (1-21) peptide substrate plus 5 µM acetyl CoA. Enzymatic reactions contained 1% DMSO.



4. Z'-factor determination.

If screening, you may wish to perform a Z'-factor determination experiment as part of your assay validation. Two conditions are chosen that will generate a high and low signal (for example, with and without a particular concentration of inhibitor, or with and without a particular concentration of cofactor). In general, a Z'-factor above 0.5 is highly desirable for screening campaigns.

lance_epi_zprime_ASK.jpg
Figure 8. p300 (0.1 nM) was pre-incubated with or without 100 µM anacardic acid for 10 min. Enzymatic reactions were initiated by the addition of 200 nM biotinylated H3 (1-21) peptide substrate plus 5 µM acetyl CoA and stopped after 30 minutes. Enzymatic reactions contained 1% DMSO.


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Tips and FAQs


  • Acetyltransferase and methyltransferase recombinant enzymes can exhibit low activity in biochemical assays, compared to other types of enzymes such as kinases. Reaction buffer optimization (including pH, salt surfactant concentration, etc.), enzyme titration, and time-course experiments are critical in achieving a good signal window.
  • In order to avoid stability issues and minimize assay variability, enzyme, cofactor/s, substrate/s, and test compounds should be diluted in assay buffer just before use.
  • Acetyl CoA cofactor is prepared at 50 mg/mL (56.9 mM) in h3O, aliquoted and stored at -80 °C. PerkinElmer R&D obtains acetyl CoA from Sigma (#A2056).
  • S-adenosylmethionine is prepared at 30 mM in 5 mM h3SO4/10% ethanol (v/v) in h3O, aliquoted, and stored at -80 °C. PerkinElmer R&D obtains S-adenosylmethionine from Sigma (#A7007).
  • The concentrations of biotin-peptide substrate indicated in the Tech Notes are the best concentrations to work with for the model enzymes indicated. For other enzymes, or if you would like to use a different peptide (longer, shorter, bearing other modifications already, etc.), it is possible that you will need to adjust the substrate concentration. Nevertheless, we suggest that you initially use the substrate concentration indicated on the technical notes (for a given anti-mark antibody) to generate your enzyme progression curves. You should only start adjusting the biotin-peptide substrate concentration if the enzymatic assay does not work well and other components in the assay have been optimized already. If possible, we strongly advise that you try changing the concentration of the co-factor or enzyme, or the reaction time rather than changing the concentration of biotin-peptide substrate. An important thing to keep in mind is that, generally, the concentration of biotin-peptide substrate cannot be increased too much because this could cause the detection assay to “hook” on the biotin-streptavidin interaction site.

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Tech notes


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Custom reagents and custom assay development at PerkinElmer


PerkinElmer offers custom Europium labeling and ULight dye labeling services as well as custom assay development. If you are interested in custom services, please contact our custom teams:

ON>POINT® Custom Labeling and Conjugation Services
ON>POINT® Custom Assay Development Services

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