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The Importance of Histone Modifications
The addition or removal of modifications such as phospho, methyl and acetyl functional groups to histones has a profound effect on the regulation of transcription, chromosome packaging and DNA damage repair. Screening extracts for specific histone modifications is also a simple way to assess cell health and compound effects. Active Motif offers a variety of high-throughput, quantitative, ELISA-based assays to measure global changes in histone modifications in response to drug treatment.
Histone Modification ELISAs for:
Active Motif’s comprehensive selection of Histone Modification ELISAs provides a simple solution for screening changes in histone H3 modification levels from purified core histones or histones isolated by acid extraction. These assays are:
- QUANTITATIVE - Includes control standards for easy and accurate quantitation
- HIGH THROUGHPUT - Uses a stripwell microplate format
- SIMPLE – ELISA based colorimetric assay is easy to read
- SPECIFIC – Has been tested for cross-reactivity with other histone modifications
In addition to histone tail modifications, the specific variant of histone protein incorporated into chromatin can serve as an epigenetic indicator of cell health. See our Nu.Q® H3 and Nu.Q® H3.1 Assays for more information.
To learn more about our Histone Modification ELISAs, click on the Documents, Background, or Publications tabs below.
What are Histone Modifications?
Epigenetic modifications such as phosphorylation, acetylation and methylation at specific amino acid residues on the histone tails influence higher-order chromatin structure that regulates the nuclear processes of transcription, chromosome packaging and DNA damage repair1. Many of these specific histone modifications are conserved throughout eukaryotes. While the biological significance of some histone modifications remains to be understood, some have been demonstrated to correlate very closely with specific cellular states like transcriptional activity2,3.
Histone methylation is a post-translational modification that occurs on arginine and lysine residues and is catalyzed by histone methyltransferase enzymes belonging to either the PRMT1, SET-domain or DOT1/DOT1L protein families4. The regulational consequence of histone methylation on transcriptional activation or repression depends on the site and degree of methylation. Histone lysine methylation occurs on histones H3 and H4 in either a monomethyl, dimethyl or trimethyl state.
Trimethylation of lysine 4, lysine 36 or lysine 79 of histone H3 (H3K4, H3K36 or H3K79) is generally associated with euchromatin and transcriptional activity. Conversely, trimethylation of lysine 9 or lysine 27 of histone H3 (H3K27) and trimethylation of lysine 20 on histone H4 (H4K20) are correlated with heterochromatin formation and transcriptional repression. Histone methylation, however, is not a permanent state. Histone demethylases belonging to the LSD1 or Jumonji domain-containing (Jmjc) families can remove methyl groups and change transcriptional activity.
Histone phosphorylation is a post-translational modification that occurs at serine 10 (Ser10), serine 28 (Ser28), threonine 3 (Thr3) and threonine 11 (Thr11) on histone H3 during mitosis5,6. Phosphorylation of serine 10 and serine 28 on the tail of histone H3 occur early in mitosis when chromosomes begin to condense and during premature chromosome condensation induced in S-phase cells. Histone H3 is phosphorylated on serine 10 (Ser10) during late S phase or G2 phase, while the phosphorylation of serine 28 (Ser28) occurs during prophase7,8. In contrast to serine 10, phosphorylation of serine 28 has never been observed in interphase7. Histone phosphorylation at serine 10 and serine 28 serve as important markers for cells undergoing mitosis and also show the importance of histone modifications during the cell cycle.
Histone acetylation is a post-translational modification that effects the nucleosome structure and therefore the ability of transcription factors to access the DNA and regulate gene expression. Histone acetylation, therefore, is an important epigenetic tag within chromatin. Hyperacetylation of histones has been associated with a relaxed chromatin structure (euchromatin) and active gene transcription, while hypoacetylation of histones can lead to transcriptional repression by condensing the structure of chromatin (heterochromatin) and restricting the access of transcription factors to the DNA9.
Histone acetylation is a reversible process. Two superfamilies of enzymes are involved in histone acetylation: histone acetyltransferases (HAT) and histone deacetylases (HDAC), which catalyze the addition and removal of acetyl groups on histones. Histone acetyltransferases (HAT) are enzymes that catalyze the acetylation of lysine residues in the N-terminal tails of histone proteins. The source of the acetyl group in histone acetylation is Acetyl-Coenzyme A. In addition to histones, some HAT enzymes also target non-histone protein substrates, such as transcription factors (i.e. p53) and structural proteins (i.e. α-tubulin). Histone deacetylases remove the acetyl group from the histone and transfer it to Coenzyme A.
Sandwich ELISAs for Histone H3 Modifications
Histone Modification ELISAs provide a simple, sensitive method for detecting changes in specific histone modifications from purified core histones or histones isolated by acid extraction. Active Motif has applied our expertise in making histone modification antibodies to produce optimal antibody pairs for the detection of specific histone modifications in a sandwich ELISA format. These kits are sandwich ELISAs that utilize a capture antibody against histone H3 and a primary antibody specific for the modification of interest. A secondary antibody conjugated to horseradish peroxidase (HRP) and developing solutions provide a sensitive colorimetric readout that is easily quantified by spectrophotometry. The assay is performed in a convenient 96-stripwell plate, enabling low or high throughput screening.
Included in each Histone Methylation ELISA is a methylated recombinant histone protein made using Active Motif's patented technology. Each Histone Acetyl ELISA includes an acetylated recombinant histone protein. These included proteins can be used to build a reference standard curve to quantitate the amount of specifically methylated or acetylated histone H3 in your samples. Click on our link to get a complete list of our Recombinant Histones and Modified Histones. The Total Histone H3 ELISA includes an unmodified Recombinant Histone H3 protein. Each Histone Phosphorylation ELISA contains treated and untreated acid extracts for use as a positive and negative controls.
Histone Modification ELISA Advantages
- Increased sensitivity over immunoblotting methods
- Works with purified core histones or histones isolated by acid extraction from tissue or cell samples
- Results in less than three hours
- Specific antibody detection ensures low background and no cross-reactivity with other modifications
- Colorimetric readout enables easy, quantitative analysis with spectrophotometry at 450 nm
- 96-stripwell format enables both high and low throughput
- Positive controls included in each kit
- Bartova, E. et al. (2008) J. Histochemistry & Cytochemistry 56:711-721.
- Kirmizis, A. et al. (2004) Genes & Dev. 18:1592-1605.
- Squazzo, S. et al. (2006) Genome res. 16:890-900.
- Martin, C. & Zhang, Y. (2005) Nature 6:838-849.
- Preuss, U. et al. (2003) Nucleic Acids Res. 31:878-885.
- Bonenfant, D. et al. (2007) Mol. & Cellular Proteomics 6(11):1917-1932.
- Goto, H. et al. (1999) J. Biol Chem. 274(36):25543-25549.
- Hooser, A.V. et al. (1998) J. of Cell Science 111:3497-3506.
- Wang, L. et al. (1997) Mol. and Cell. Biol. 17(1):519-527.