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A challenge in studying epigenetic enzymes and their corresponding modifications is their existence in multiple protein complexes and the complexes’ impact on their activity.  Therefore, the ability to study these enzymes and to identify compounds that translate into useful drugs in vivo is greatly facilitated by the ability to incorporate cell-based assays using therapeutically relevant cell backgrounds. 

Our BacMam Histone H3 Cellular Assay Kits allow you to choose the modification that corresponds to your enzyme of interest in your chosen cell background or multiple cell backgrounds.  

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Assay Principle

The BacMam reagent is used to transduce your cells and drives expression of GFP-Histone H3 fusion protein in your cell background of interest. The GFP-Histone H3 is now a substrate for a variety of epigenetic enzymes such as methyltransferases.  In the presence of a terbium (Tb)-labeled modification-specific antibody (such as anti histone H3K4me2), the binding of the antibody to the Histone H3 substrate results in time-resolved fluorescence resonance energy transfer (TR-FRET) between the GFP and the Tb.

Monitoring the TR-FRET readout in the presence of cells treated with compounds enables the identification of inhibitors of epigenetic enzymes for that particular modification.

Figure 1. Assay overview. Cells are mixed with BacMam Reagent encoding GFP-tagged Histone H3 protein and plated onto a 384-well assay plate. Cells are left untreated or treated with compound for 20 to 24 hours. Cells are lysed in the presence of a terbium (Tb)-labeled modification-specific antibody and TR-FRET is detected using a fluorescence microplate reader with standard TR-FRET settings. 

Assay Benefits

The BacMam Histone H3 Cellular Assays combine the portability of BacMam with LanthaScreen® technology for detection of epigenetic modifications of Histone H3 within the native cellular environment.

Key Benefits for Epigenetics Researchers:
  • Identify more relevant inhibitors since the epigenetic enzymes are in their natural protein complexes
  • Use your cell background of choice (including primary and stem cells) with the portability of BacMam
  • Screen quickly and conserve precious cell sample with a simple and homogenous high-throughput screening (HTS) – amenable assay format
  • Quickly develop assays in less than one week without having to generate stable cell lines
  • Measure multiple modifications, such as phosphorylation, acetylation, or methylation by simply switching out the antibody
  • Improve data quality with the advantages of TR-FRET that provides reduced data noise due to the ratiometric readout

 Learn more about:

  • BacMam-enabled Cellular Assays
  • The LanthaScreen™ Cellular Assay Technology

 

H3K27me3 Detection in Various Cell Types

 

Figure 1. Detecting Histone H3 Lysine 27 trimethylation in various cell backgrounds. BacMam enables portability of the assays to relevant cell backgrounds.  Here, BacMam Histone H3 Reagent was added to a panel of different cell types to achieve the indicated virus concentration (v/v). Subsequent addition of the LanthaScreen® Tb-anti-Histone H3K27me3 Antibody enabled detection of TR-FRET signals showing a range of assay windows depending on the cell background.  The differences in assay window can be attributed to such factors as variations in the methylation status of histone H3 and/or the transduction efficiency of the cell background.

 

 

 

EZH2 RNAi Decreases H3K27me3 Levels

 

Figure 1. siRNA knockdown analysis shows EZH2 RNAi decreases H3K27me3 levels in HeLa cells. Stealth RNAi oligos were used to analyze the contribution of four different methyltransferase to histone H3 trimethylation of lysine 27 in HeLa cells. Using 15% BacMam Histone H3 Reagent, RNAi knockdown of H3K27me3 was most significant for EZH2 as expected.

 

Histone methyltransferase Histone H3 lysine modifications
 SETD7 (SET7/9) H3K4me1
 SMYD3 H3K4me2 and H3K4me3
 EHMT2 (G9a) H3K9me1 and H3K9me2
 EZH2

 H3K27me1, K27me2, K27me3

Table 1. Enzymes and their corresponding modifications.

Assay Principle

Assay Principle

The BacMam reagent is used to transduce your cells and drives expression of GFP-Histone H3 fusion protein in your cell background of interest. The GFP-Histone H3 is now a substrate for a variety of epigenetic enzymes such as methyltransferases.  In the presence of a terbium (Tb)-labeled modification-specific antibody (such as anti histone H3K4me2), the binding of the antibody to the Histone H3 substrate results in time-resolved fluorescence resonance energy transfer (TR-FRET) between the GFP and the Tb.

Monitoring the TR-FRET readout in the presence of cells treated with compounds enables the identification of inhibitors of epigenetic enzymes for that particular modification.

Figure 1. Assay overview. Cells are mixed with BacMam Reagent encoding GFP-tagged Histone H3 protein and plated onto a 384-well assay plate. Cells are left untreated or treated with compound for 20 to 24 hours. Cells are lysed in the presence of a terbium (Tb)-labeled modification-specific antibody and TR-FRET is detected using a fluorescence microplate reader with standard TR-FRET settings. 

Assay Benefits

Assay Benefits

The BacMam Histone H3 Cellular Assays combine the portability of BacMam with LanthaScreen® technology for detection of epigenetic modifications of Histone H3 within the native cellular environment.

Key Benefits for Epigenetics Researchers:
  • Identify more relevant inhibitors since the epigenetic enzymes are in their natural protein complexes
  • Use your cell background of choice (including primary and stem cells) with the portability of BacMam
  • Screen quickly and conserve precious cell sample with a simple and homogenous high-throughput screening (HTS) – amenable assay format
  • Quickly develop assays in less than one week without having to generate stable cell lines
  • Measure multiple modifications, such as phosphorylation, acetylation, or methylation by simply switching out the antibody
  • Improve data quality with the advantages of TR-FRET that provides reduced data noise due to the ratiometric readout

 Learn more about:

  • BacMam-enabled Cellular Assays
  • The LanthaScreen™ Cellular Assay Technology

 

H3K27me3 in Various Cell Types

H3K27me3 Detection in Various Cell Types

 

Figure 1. Detecting Histone H3 Lysine 27 trimethylation in various cell backgrounds. BacMam enables portability of the assays to relevant cell backgrounds.  Here, BacMam Histone H3 Reagent was added to a panel of different cell types to achieve the indicated virus concentration (v/v). Subsequent addition of the LanthaScreen® Tb-anti-Histone H3K27me3 Antibody enabled detection of TR-FRET signals showing a range of assay windows depending on the cell background.  The differences in assay window can be attributed to such factors as variations in the methylation status of histone H3 and/or the transduction efficiency of the cell background.

 

 

 

EZH2 RNAi Data

EZH2 RNAi Decreases H3K27me3 Levels

 

Figure 1. siRNA knockdown analysis shows EZH2 RNAi decreases H3K27me3 levels in HeLa cells. Stealth RNAi oligos were used to analyze the contribution of four different methyltransferase to histone H3 trimethylation of lysine 27 in HeLa cells. Using 15% BacMam Histone H3 Reagent, RNAi knockdown of H3K27me3 was most significant for EZH2 as expected.

 

Histone methyltransferase Histone H3 lysine modifications
 SETD7 (SET7/9) H3K4me1
 SMYD3 H3K4me2 and H3K4me3
 EHMT2 (G9a) H3K9me1 and H3K9me2
 EZH2

 H3K27me1, K27me2, K27me3

Table 1. Enzymes and their corresponding modifications.

For Research Use Only. Not for use in diagnostic procedures.