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Epigenetics & Chromatin

Histone H3 acetyl Lys9 ELISA (H3K9)

to quantitate the level of histone H3 lysine 9 acetylation

Histone Modification ELISAs provide a simple, sensitive method for detecting changes in histone acetylation levels from purified core histones or histones isolated by acid extraction. For added convenience and a more quantitative interpretation of results, the Histone Modification ELISAs also include acetylated recombinant histone protein for use as a reference standard curve. For complete details, click the ELISA Method tab below.

Histone H3 lysine 9 acetylation (H3K9) has been correlated with transcriptional activation and chromatin reassembly during DNA replication and can occur in response to mitogenic and hormonal stimulation of cells. The Histone H3 acetyl Lys9 ELISA is a sandwich ELISA for screening the levels of histone H3 acetyl Lys9 in your samples. The detecting antibody is specific for acetyl Lys9 and will not cross-react with other modifications, enabling small changes in acetylation levels to be detected. Click on the H3K9 Info tab below for data and more information.

 
Name Format Cat No. Price  
Histone H3 acetyl Lys9 ELISA 1 x 96 rxns 53114 $525 Buy Now

Histone H3 acetylated Lys9 (H3K9)

The acetylation of lysine 9 on histone H3 (H3K9) has been correlated with the regulation of gene transcription, making acetylated lysine 9 on histone H3 a significant marker in studying the state of transcription activity. Acetylation of histone H3 at lysine 9 is linked to corresponds to transcriptional activation and occurs in response to mitogenic and hormonal stimulation in mammalian cells. In addition to its involvement in regulation of transcriptional activity, acetylation of lysine 9 also plays a critical role in regulating histone H3 recruitment to the newly assembled chromatin complex during DNA replication.

Histone H3 acetyl Lys9 ELISA (H3K9ac)

The Histone H3 acetyl Lys9 ELISA is capable of detecting the level of histone H3 acetylated at lysine 9 from core histone preparations or histones purified by acid extraction from tissue or cell samples (Figure 1). The Histone H3 acetyl Lys9 sandwich ELISA assay is also tested for cross-reactivity with other site and degree-specific recombinant proteins as shown in Figure 2.

 

Histone H3 acetyl Lys9 ELISA
 
Figure 1: Histone H3 acetyl Lys9 ELISA (H3K9ac).

The Histone H3 acetyl Lys9 ELISA was used to assay  HeLa acid extracts (50 ng) and purified HeLa core histones (50 ng) made using Active Motif's Histone Purification Mini Kit (Catalog No. 40026). The provided Recombinant Histone H3 acetyl Lys9 protein was assayed from 0.039 - 2.5 ng/well as a reference standard curve. Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

 

Specificity of Histone H3 acetyl Lys9 ELISA
 
Figure 2: Specificity of the Histone H3 acetyl Lys9 ELISA (H3K9ac).

Recombinant Histone H3, mono-, di- and trimethyl Lys9, acetyl Lys9 and acetyl Lys14 proteins were assayed from 0.156 ng - 10 ng per well using the Histone H3 acetyl Lys9 ELISA. These results indicate the specificity of the assay. There is extremely low background from histone H3 and no cross-reactivity with methylated Lys9 or acetylated Lys14. This means that small, specific changes in acetyl Lys9 levels can easily be detected with this kit.

The Histone Modification ELISA Advantage

Historically, screening for histone modifications from sample preparations have been conducted using immunoblotting and chromatin immunoprecipitation methods, which can be time-consuming, do not allow for high-throughput and provide only semi-quantitative results. Active Motif's Histone Modification ELISAs provide a fast, sensitive assay with the flexibility to screen from 1 to 96 samples in a single experiment.

Included in each Histone Acetylation ELISA is an acetylated recombinant histone protein made using Active Motif's patented protein synthesis technology.  Likewise, included in each Histone Methylation ELISA is a methylated recombinant histone protein made using Active Motif's patented protein synthesis technology. These included proteins can be used to build a reference standard curve to quantitate the amount of either specifically acetylated or methylated histone H3 (respectively) in your samples. Click on our link for more information on our patented protein synthesis technologies and 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.

Why use the Histone Methylation, Histone Acetylation and Histone Phosphorylation ELISAs?

  • Increased sensitivity over immunoblotting methods
  • 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

The Histone Acetylation ELISA Method

The Histone ELISAs to detect acetylation levels on histone H3 are sandwich ELISAs that utilize a monoclonal histone H3 antibody to capture histone H3 from purified core histones or histones isolated by acid extraction from tissue or cell samples. A polyclonal antibody specific for the modification of interest is used for detection, while a secondary antibody conjugated to horseradish peroxidase (HRP) and developing solutions provide a sensitive colorimetric readout that is easily quantified by spectrophotometry at 450 nm. The assay is performed in a convenient 96-stripwell plate, which enables either low or high throughput screening.

The Histone Methylation ELISA Method

The Histone ELISAs to detect site- and degree-specific lysine methylation levels on histone H3 are sandwich ELISAs that utilize a monoclonal histone H3 antibody to capture histone H3 from purified core histones or histones isolated by acid extraction from tissue or cell samples. A polyclonal antibody specific for the modification of interest is used for detection, while a secondary antibody conjugated to horseradish peroxidase (HRP) and developing solutions provide a sensitive colorimetric readout that is easily quantified by spectrophotometry at 450 nm. The assay is performed in a convenient 96-stripwell plate, which enables either low or high throughput screening.

The Histone Phosphorylation ELISA Method

The Histone ELISAs to detect phosphorylation levels of serine 10 (pSer10) or serine 28 (pSer28) on histone H3 are sandwich ELISAs that utilize a C-terminal histone H3 antibody to capture histone H3 from purified core histones or histones isolated by acid extraction from tissue or cell samples. A monoclonal antibody specific for the phosphorylation site of interest is used for detection, while a secondary antibody conjugated to horseradish peroxidase (HRP) and developing solutions provide a sensitive colorimetric readout that is easily quantified by spectrophotometry at 450 nm. The assay is performed in a convenient 96-stripwell plate, which enables either low or high throughput screening.

Flow Chart of Histone Modification ELISA Method

Flow chart of the histone modification elisa
 
Figure 1: Flow chart of the Histone Modification ELISA Method.

Histone Modification ELISAs utilize a histone H3 antibody to capture histone H3 from samples and a modification specific antibody for detection. A secondary antibody conjugated to horseradish peroxidase and developing solutions provide a colorimetric readout that is quantified by spectrophotometry. (Click image to enlarge.)

Contents & Storage

Histone H3 acetyl Lys9 ELISA Kits include a 96-stripwell Histone H3 Capture plate, a primary antibody specific for Histone H3 acetyl Lys9, HRP-conjugated anti-rabbit IgG secondary antibody, Assay Dilution Buffer, 20X Wash Buffer, Developing Solution, Stop Solution, Recombinant Histone H3 acetyl Lys9 positive control protein and plate sealer. Storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.