Histone H3 methylated Lys9 ELISA (H3K9)

Histone H3 methylated Lys9 (H3K9)

The methylation of lysine 4 on histone H3 (H3K9) has been correlated with the regulation of gene transcription, making methylated lysine 9 on histone H3 a significant marker in studying the state of transcription activity. Methylation of histone H3 at lysine 9 is linked to heterochromatin formation and transcriptional repression. Histone methyltransferases G9a, G9a-related protein (GLP) and SUV39H1 are believed to have specificity for methylation of lysine 9 on histone H3, but only SUV39H1 is able to recruit the chromodomain-containing heterochromatin protein 1 (HP1) to chromatin.

Histone H3 trimethyl Lys9 ELISA (H3K9me3)

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

Figure 1: Histone H3 trimethyl Lys9 ELISA (H3K9me3).

The Histone H3 trimethyl Lys9 ELISA was used to assay purified HeLa core histones (250 - 500 ng) made using Active Motif's Histone Purification Mini Kit (Catalog No. 40026) and HeLa acid extracts (5 - 10 µg). The provided Recombinant Histone H3 trimethyl Lys9 protein was assayed from 7.8125 - 500 ng/well as a reference standard curve. Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

Figure 2: Specificity of the Histone H3 trimethyl Lys9 ELISA (H3K9me3).

Recombinant Histone H3, mono-, di- and trimethyl Lys9 proteins were assayed from 15 ng - 1 ng per well using the Histone H3 trimethyl Lys9 ELISA. These results indicate the specificity of the assay. There is extremely low background from histone H3 and little cross-reactivity for mono- or dimethyl Lys9. This means that small, specific changes in trimethyl 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 Methylation ELISA is a methylated recombinant histone made using Active Motif's patented protein synthesis technology. This protein can be used to build a reference standard curve to quantitate the amount of specifically methylated histone H3 in your samples. 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 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 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

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 trimethyl Lys9 ELISA Kits include a 96-stripwell Histone H3 Capture plate, a primary antibody specific for Histone H3 trimethyl Lys9, HRP-conjugated anti-rabbit IgG secondary antibody, Assay Dilution Buffer, 20X Wash Buffer, Developing Solution, Stop Solution, Recombinant Histone H3 trimethyl 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.