Active Motif,
Tools to analyze nuclear function,
Your CartYour Cart 0 items

STAY INFORMED

Sign up to receive new product updates
and promotional pricing.

Epigenetics & Chromatin

Histone H3 PTM Multiplex Kit

simultaneously interrogate multiple histone H3 PTMs

Active Motif's Histone H3 PTM Multiplex Assay is a novel high throughput bead-based ELISA assay designed for use with the MAGPIX®, Luminex® 200™ or FLEXMAP 3D® instruments to enable simultaneous interrogation of the levels of multiple histone H3 PTMs within the same sample. The 3-hour assay is ideally suited for high-throughput screening and profiling of histone modification levels of clinical or compound-treated samples. The multiplexing ability means you can simultaneously screen for on-target and off-target effects within the same sample. This kit offers several advantages over traditional methods (e.g. Western Blotting) including faster turnaround times and the ability to utilize smaller sample amounts.


Histone PTM Services RequestServices Request Button

Are you interested in having Active Motif provide this as a service for you?

 


Histone PTM Multiplex Assay Principle
Schematic of the Histone H3 PTM Multiplex Assay to study Histone H3 PTMs levels.

The 96-well plate-based assay works with nanogram quantities per well of acid extracted lysates or purified histones. The inclusion of a Histone H3 Total antibody bead set into the multiplex reaction enables normalization of Histone PTM levels across samples. To learn more about how the Histone PTM Multiplex Assay works or to read about the Luminex* technology, click on the Method tab below. To view data highlighting the advantages of the Histone PTM Multiplex Assay and its application, click on the Data tab. To view manuals, datasheets and other related documents, click on the Documents tab.

To download a blank 96-well plate template to help with plate set-up, CLICK HERE.

*Luminex is a registered trademark of the Luminex Corporation. Users of the assay products are agreeing to the Luminex terms and conditions as stated in the product manual.

 
Name Format Cat No. Price  
Histone H3 PTM Multiplex Kit 96 rxns 33115 $425 Buy Now
Histone H3 Total Ab-conjugated Beads 48 rxns 33116 $180 Buy Now
Histone H3 pan-acetyl Ab-conjugated Beads 48 rxns 33123 $180 Buy Now
Histone H3K4me3 Ab-conjugated Beads 48 rxns 33121 $180 Buy Now
Histone H3K9ac Ab-conjugated Beads 48 rxns 33117 $180 Buy Now
Histone H3K9me1 Ab-conjugated Beads 48 rxns 33118 $180 Buy Now
Histone H3K9me2 Ab-conjugated Beads 48 rxns 33119 $180 Buy Now
Histone H3K9me3 Ab-conjugated Beads 48 rxns 33120 $180 Buy Now
Histone H3S10ph Ab-conjugated Beads 48 rxns 33122 $180 Buy Now
Histone H3T11ph Ab-conjugated Beads 48 rxns 33128 $180 Buy Now
Histone H3K27ac Ab-conjugated Beads 48 rxns 33127 $180 Buy Now
Histone H3K27me2 Ab-conjugated Beads 48 rxns 33124 $180 Buy Now
Histone H3K27me3 Ab-conjugated Beads 48 rxns 33125 $180 Buy Now
Histone H3K36me3 Ab-conjugated Beads 48 rxns 33129 $180 Buy Now
Histone H3K56ac Ab-conjugated Beads 48 rxns 33126 $180 Buy Now

Histone Post-translational Modifications (PTMs)

Histones are responsible for packaging DNA into nucleosomes, the basic structural unit of chromatin. They are subject to a variety of post-translational modifications on the N-terminal tails of histone proteins, including acetylation, methylation and phosphorylation, that function to regulate transcription, chromosome packaging and DNA damage repair1-10. Many of these specific histone modifications are conserved throughout eukaryotes. These histone modifications are recognized and bound by specific proteins that are coined 'readers', 'writers', and 'erasers'11.

The importance of studying histone modifications has implications for human health and disease since a strong correlation exists between specific histone modifications and human pathologies. The diseased state is associated with altered epigenetic profiles of DNA methylation or histone modification patterns. Numerous publications have described cancer-specific changes in histone modification levels12-22. Relative to DNA methylation, far less is known about the biological significance of histone PTMs, mostly due to limitations in available technologies.

Current methods to study histone PTMs included Western blot, immunohistochemistry and genome-wide mapping of histone PTM binding sites. All of these methods are time-consuming and lack high throughput capabilities. To address these issues, Active Motif has partnered with Luminex®, the industry leader in multiplexing technology, to develop the first multiplex epigenetic assay for the study of histone H3 PTMs. This assay enables high throughput processing using nanogram quantities of sample to interrogate multiple histone modifications within a single well. The assay format also offers the ability to normalize data against total histone H3 levels to evaluate relative histone H3 PTM levels across different samples.

How Do Histone H3 PTM Multiplex Kits Work?

The Histone H3 PTM Multiplex Assays work as bead-based "sandwich" assays to interrogate the levels of histone H3 modifications within acid extracted cell lysates or purified histones (see Figure 1). Samples are diluted in Assay Buffer and added to a 96-well assay plate. Histone post-translational modification antibodies, which are coupled to color-coded magnetic beads, are added to each well to capture the N-terminal histone H3 PTMs of interest in the sample. Using a 96-well magnetic plate, wells are washed to remove any unbound sample. A biotinylated Histone H3 antibody is added as a reporter to recognize the C-terminal domain of the histone samples bound to the antibody-conjugated beads. Following a wash step to remove unbound biotinylated antibody, streptavidin-phycoerythrin (SA-PE) is added to each well to bind the biotinylated reporter antibody. A final wash is performed to remove unbound SA-PE and the beads are resuspended in Wash Buffer and read using a MAGPIX™, Luminex® 200™ or FLEXMAP 3D™ instrument and xPONENT® software. To view amanual for the Histone H3 PTM Multiplex assay, datasheets and other related documents, click on the Documents tab above.

Since each antibody-conjugated bead contains a different fluorescent label, antibodies for multiple histone PTMs can be added to the same sample for multiplexing. The readout from the streptavidin-phycoerythrin is proportional to the amount of histone PTM captured. A Histone H3 Total Ab-conjugated Bead set is available for normalization of PTM data across different samples. Active Motif offers the Histone H3 PTM Multiplex Kit and Histone Antibody-conjugated Beads to create your own custom multiplex or singleplex assays. All antibody-conjugate bead sets have been confirmed to work when multiplexed without any negative effects on assay results. A universal Assay Positive Control is also included to monitor the consistency of the performance of the assay between runs.

Luminex Histone PTM Multiplex Assay Principle
Figure 1: Histone H3 PTM Multiplex Kits are bead-based sandwich assays to study histone H3 PTMs levels.

 

The Luminex Principle

The Luminex technology is a bead-based assay system using tiny, 6.4 micron superparamagnetic beads. Each bead contains its own distinct dye ratio which will generate a unique fluorescence pattern to enable individual bead identity. Active Motif provides the beads conjugated to histone antibodies which can be used individually or in combination to capture the N-terminal histone modification(s) of interest from the sample. Once the beads are identified by their distinct fluorescence within the Luminex instrument, the identity of the captured histone PTM is also known. A biotinylated Histone H3 antibody is added as a reporter to recognize the C-terminal domain of the bound histone samples. Streptavidin-phycoerythrin will bind to the biotinylated Histone H3 antibody and produce a fluorescence signal proportional to the amount of histone binding.

If using the Luminex® 200™ instrument, the beads are individually passed through a small shaft where a light source excites the internal dye that is used to identify each bead. Based on the dye ratios, each bead will exhibit a unique fluorescence that can be identified with a unique bead region. A second wavelength is used to determine the magnitude of the streptavidin-phycoerythrin signal (see Figure 2). A minimum of 100 beads are read for each well of the assay. If using the MAGPIX® instrument, beads are collected as a monolayer on the sample chamber. LEDs are used to illuminate the chamber and CCD imaging technology capture a series of images. The software analyzes the images to determine the bead identity and phycoerythrin signal. The Luminex xPONENT® software program will provide a real-time readout of signal measured as median fluorescent intensity (MFI).

Luminex 200 instrument reading
Figure 2: The Luminex® 200™ instrument uses two distinct wavelengths to analyze individual beads as they pass through the machine for bead identity and phycoerythrin signal.

 

What's in the Box?

Each Multiplex Kit contains a 96-well assay plate, buffers, Biotinylated Histone H3 Antibody, a universal Assay Positive Control and streptavidin-phycoerythrin for detection. Antibody-conjugated beads must be purchased separately to complete the assay. The individual bead packaging allows researchers to customize their assays for singleplex or multiplex analysis simply by selecting antibody-conjugated beads for their PTM of interest. 

Note: A complete assay requires the purchase of both the Histone H3 PTM Multiplex Kit containing the buffers and the Histone antibody-conjugated bead(s) of interest.

 

References

  1. Kirmizis, A., et al. (2004) Genes & Dev, 18: 1592-1605.
  2. Squazzo, S., et al. (2006) Genome Res, 16: 890-900.
  3. Kouzarides T. (2007) Cell, 128 (4): 693-705.
  4. Lee J.S., et al. (2010) Cell, 142 (5): 682-685.
  5. Berger. S.L., et al. (2007) Nature, 447 (7143): 407-412.
  6. Sims, R.J. & Reinberg, D. (2008) Nat Rev Mol. Cell Biol, 9 (10): 815-820.
  7. Millar, C.B., et al. (2006) Genes & Dev, 20: 711-722.
  8. Doyon, Y., et al. (2006) Mol Cell, 21: 51-64.
  9. Qin, S., Parthun, M.R. (2006) Mol Cell Biol, 26: 3649-3658.
  10. Shrogen-Knaak, M., et al. (2006) Science, 311: 844-847.
  11. Jenuwein, T. & Allis, C.D. (2001) Science, 293 (5532): 1074-1080.
  12. Bianco-Miotto, T., et al. (2010) Cancer Epidemiol Biomarkers Prev, 19(10): 2611-2622.
  13. Ellinger, J., et al. (2010) Int J Cancer, 127(10): 2360-2366.
  14. Ellinger, J., et al. (2010) Prostate, 70(1): 61-69.
  15. Elsheikh, S.E., et al. (2009) Cancer Res, 69(9): 3802-3808.
  16. Fraga, M.F., et al. (2005) Nat Genet, 37(4): 391-400.
  17. I, H., et al. (2010) Cancer Epidemiol Biomarkers Prev, 19(2): 566-573.
  18. Manuyakorn, A., et al. (2010) J Clin Oncol, 28(8): 1358-1365.
  19. Mosashvilli, D., et al. (2010) Cancer Sci, 101(12): 2664-2669.
  20. Seligson, D.B., et al. (2009) Am J Pathol, 174(5): 1619-1628.
  21. Seligson, D.B., et al. (2005) Nature, 435(7046): 1262-1266.
  22. Zhou, L.X., et al. (2010) Aian J Androl, 12(2): 171-179.

Histone H3 PTM Multiplex Assay offers higher throughput and better sensitivity than Western blot

Comparison of Histone H3S10ph in WB versus Histone PTM Multiplex Kit
Figure 1: Comparison of data obtained from a Western blot or Histone H3 PTM Multiplex Assay for amounts of H3S10ph.

To evaluate changes in total Histone H3 and H3S10ph levels from untreated and colcemid treated HeLa acid extract, a Western blot (left image) was compared with a 2-plex Histone H3 PTM assay (right image) using the Total H3 and H3S10ph Antibody-conjugated Bead sets. The Western blot lacks throughput capabilities, requires microgram sample quantities to study each antibody and does not provide normalized information. The Histone H3 PTM Multiplex assay requires only nanogram quantities of sample from which multiple antibodies can be evaluated simultaneously in a single well. Data can then be normalized against the Total H3 values to demonstrate the relative amounts of H3S10ph in each sample.


Analyze multiple Histone H3 PTMs in multiplex in a single well

13 plex analysis of Histone PTMs
Figure 2: Histone H3 PTM Multiplex Kit using 13 bead sets in multiplex.

H13 Histone Antibody-conjugated Beads were tested in multiplex on 0.5 μg Hela acid extract. Results show MFI values generated using 1:250 Biotinylated Histone H3 reporter antibody for detection. Raw MFI values are displayed as the average of duplicate wells. The combination of all 13 bead sets within a single well had no discernible effects on the assay.


Sensitive assay requires only nanogram quantities of sample to detect PTMs

Dynamic Range of Histone H3K9ac Bead set
Figure 3: Dynamic Range of the Histone H3K9ac Ab-conjugated Beads.

An example of the dynamic range of the Histone H3K9ac Ab-conjugated Beads is shown for both acid extracted HeLa lysate and core histones that were purified from HeLa cells using Active Motif's Histone Purification Mini Kit (Catalog No. 40026). The Histone H3 PTM Multiplex Assay is capable of detecting H3K9ac modifications from both sample types using only nanogram quantities, but the purified histones show a much larger dynamic range and require less sample material per well for robust Median Fluorescent Intensity (MFI) values.


Simultaneously screen both on-target and off-target effects within the same sample

The Histone H3 PTM Multiplex assay enables rapid, high throughput screening of specific and off-target effects on histone modification levels in response to variable conditions, compound treatments or disease states. The multiplexing ability of the assay means that you can interrogate all these effects on your sample simultaneously in one well.

The Histone H3 PTM Multiplex Assay allows you to gather more information using smaller sample amounts and in less time than traditional methods to assess cellular response. The following application data demonstrate how the assay was used to generate data from dose response assessments that are consistent with previously published results in addition to previously unpublished data about off-target effects and unaffected histone marks. The assay can be completed in 3 hours and comes in a 96-well plate-based format, which means all this data was generated in a fraction of the time of conventional methods, including flow cytometry, cytotoxicity assays, HDAC assays and Western blot that were utilized to generate published data.

 

The Histone H3 PTM Assay reveals both specific and off-target effects of VPA treatment.
Figure 4: The Histone H3 PTM Multiplex Assay reveals previously unreported off-target effects of valproic acid (VPA) treatment.

Two thousand HeLa cells were seeded per well and pretreated with the indicated concentrations of VPA, an HDAC inhibitor that is also clinically utilized as a treatment for epilepsy, migraines and bipolar disorder. Acid extracts were prepared and approximately 1/10 of lysate was used to evaluate H3 pan-acetyl, H3K9ac, H3K4me3, H3K27me2 and H3S10ph Ab-conjugated beads in multiplex along with H3 Total beads for normalization to determine relative PTM values using the Histone H3 PTM Multiplex Assay. The data show that the levels of H3 pan-acetyl and H3K9ac MFI signals increase in response to higher VPA doses. Dashed lines represent IC50 values. IC50 values of 6.4 mM were reported for both H3K9ac and H3 pan-acetyl, respectively. IC50 values of 10 mM have previously been reported in dose response assessments of HeLa cells treated with VPA with indications of induction of cell cycle arrest and apoptosis at these doses (Han B.R. et al. (2013) Oncol Rep.). Off-target effects are also shown. Decreased levels of H3K4me3 and H3K27me2 are observed as off-target effects of VPA treatment on histone methylation. Although VPA treatment has previously been shown to result in decreased levels of H3K27me2 (Marinova Z. et al. (2011) Neuropharmacology.), the observation that VPA has a similar off-target effect on H3K4me3 has not previously been reported. No effect is observed on the levels of H3S10ph.


Rapidly screen PTM levels of variable compound treatments, conditions and disease states

Because the Histone H3 PTM Multiplex Assay enables normalization of values to compare relative histone modification levels across samples, cross-comparative analysis of clinical samples, disease models or various treatment conditions can be conducted with ease.

 

The Histone H3 PTM Assay reveals both specific and off-target effects of SAHA treatment of HeLa cells.
Figure 5: The Histone H3 PTM Multiplex Assay shows increased histone acetylation in HeLa cells in response to SAHA-mediated HDAC inhibition.

Two thousand HeLa cervical cancer cells were seeded per well and pretreated with the indicated concentrations of suberanilohydroxamic acid (SAHA), an HDAC inhibitor that is also marketed as a therapeutic compound for the treatment of cutaneous T-cell lymphoma. Acid extracts were prepared and approximately 1/10 of lysate was used to evaluate H3 pan-acetyl, H3K9ac, H3K4me3, H3K27me2 and H3S10ph Ab-conjugated beads in multiplex along with H3 Total beads for normalization to determine relative PTM values using the Histone H3 PTM Multiplex Assay. The data show that the levels of H3 pan-acetyl and H3K9ac MFI signals increase in response to higher SAHA doses. Dashed lines represent IC50 values. IC50 values of 4.0 μM and 4.6 μM were reported for H3K9ac and H3 pan-acetyl, respectively. This is consistent with previous SAHA dose response studies showing the occurrence of histone H3K9-14 hyperacetylation with reported IC50 values of 1.5 μM in HeLa cells in response to SAHA treatment (Beckers T. et al. (2007) Int. J. Cancer.). Off-target effects are also shown. Decreased levels of H3K4me3 and H3K27me2 are observed as off-target effects of SAHA treatment on histone methylation. No effect is observed on the levels of H3S10ph.


Histone H3 PTM Multiplex Assay data of SAHA-treated MCF-7 cells
Figure 6: Variable off-target effects are observed with the Histone H3 PTM Multiplex Assay in response to SAHA treatment of MCF-7 cells as compared to the above SAHA-treated HeLa cells.

MCF-7 breast cancer cells underwent the identical plating, SAHA treatment and Histone H3 PTM assay conditions as specified for HeLa cells in Figure 5. Briefly, two thousand MCF-7 cells were seeded and pre-treated with SAHA at increasing concentrations. Changes in levels of histone modifications were assessed in multiplex using the Histone H3 PTM Multiplex Assay with H3 pan-acetyl, H3K9ac, H3K4me3, H3K27me2 and H3S10ph Ab-conjugated beads along with H3 Total beads for normalization. The results reveal that similar increases in histone acetylation occur with SAHA treatment of MCF-7 cells as were observed in HeLa cells treated with SAHA (Figure 5). However, unlike HeLa cells, SAHA treatment results in an increase in H3K4me3 and a decrease in H3S10ph in MCF-7 cells with little change in H3K27me2 levels. IC50 values of 8.3 μM and 12.3 μM, shown as dashed lines, were reported for H3K9ac and H3 pan-acetyl, respectively. This is consistent with previous reports that show SAHA treatment results in lower IC50 values (IC50 = 1.5 μM) in HeLa cells compared with MCF-7 cells (IC50 = 6.7 μM) (Beckers T. et al. (2007) Int. J. Cancer.).

What's in the Box?

Each Multiplex Kit contains a 96-well assay plate, buffers, Biotinylated Histone H3 Antibody, a universal Assay Positive Control and streptavidin-phycoerythrin for detection. Antibody-conjugated beads must be purchased separately to complete the assay. The individual bead packaging allows researchers to customize their assays for singleplex or multiplex analysis simply by selecting antibody-conjugated beads for their PTM of interest. 

Note: A complete assay requires the purchase of both the Histone H3 PTM Multiplex Kit containing the buffers and the Histone antibody-conjugated bead(s) of interest.

Contents & Storage

The Histone H3 PTM Multiplex Kit contains the necessary buffers, reagents and controls to perform the assay, but it does not include any Histone Antibody-conjugated Beads. Beads must be purchased separately.

Histone H3 PTM Multiplex Kit

  • Biotinylated Histone H3 antibody; Store at -20°C
  • Deacetylase Inhibitor; Store at -20°C
  • Phosphatase Inhibitor Cocktail; Store at -20°C
  • Protease Inhibitor Cocktail; Store at -20°C
  • Assay Buffer AM3; Store at 4°C
  • 20X Wash Buffer; Store at 4°C
  • Streptavidin-PE; Store at 4°C
  • Assay Positive Control; Store at -80°C
  • 96-well assay plate; Store at RT
  • Plate sealer; Store at RT

Histone Antibody-conjugated Beads

  • Histone PTM Antibody-conjugated Beads; Store at 4°C