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. These kits are sandwich ELISAs that utilize a capture antibody against histone H3 and a primary antibody specifc 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.Histone H3 phospho Ser28 ELISA (H3S28ph) kits provide everything need to screen for phosphorylated serine 28 on histone H3 in human, mouse, hamster and bovine systems. Histone H3 phosphorylation on serine 28 (H3 pSer28) has been correlated with chromosome condensation, making the phosphorylation state of Ser28 an interesting marker for cells undergoing mitosis. The phosphorylation specific histone H3 antibody will not cross-react with phosphorylation of serine 10. To determine histone H3 serine 10 phosphorylation levels, we also offer a Histone H3 phospho Ser10 ELISA. For added convenience, the Histone phosphorylation ELISAs include paclitaxel treated and untreated acid extract for use as positive and negative controls, respectively. pS28 InformationHistone H3 phosphorylation Info Histone modifications such as phosphorylation, acetylation and methylation at specific amino acid residues on the histone tails that extend beyond the core nucleosome have been found to influence and regulate transcription, chromosome packaging and DNA damage repair. Phosphorylation of serine 10 and serine 28 in 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 (pS10) during late S phase or G2 phase, while the phosphorylation of serine 28 (pS28) occurs during prophase. In contrast to serine 10, serine 28 phosphorylation has never been observed in interphase. Histone H3 phospho Ser28 ELISA (H3S28ph) The Histone H3 phospho Ser28 ELISA is capable of detecting the level of histone H3 phosphorylated at serine 28 from core histone preparations or histones purified by acid extraction from tissue or cell samples (Figure 1).Figure 1: Histone H3 phospho Ser28 ELISA (H3 pSer28)The Histone H3 phospho Ser28 ELISA was used to assay 78 ng to 5 碌g of HeLa acid extracts that were either untreated (Catalog No. 36200) or paclitaxel treated (Catalog No. 36201). Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.ELISA MethodThe Histone Modification ELISA AdvantageHistorically, 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 protein 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 kitThe Histone Methylation ELISA MethodThe 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 MethodThe 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 MethodFigure 1: Flow chart of the Histone Modification ELISA MethodHistone 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. ContentsContents & StorageHistone H3 phospho Ser28 ELISA Kits include a 96-stripwell Histone H3 Capture plate, a primary antibody specific for Histone H3 phosphorylated at serine 28, HRP-conjugated secondary antibody, Assay Dilution Buffer, 20X Wash Buffer, Developing Solution, Stop Solution, paclitaxel treated HeLa acid extract for use as a positive control, untreated HeLa acid extract for use as a negative control 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.