The Pre-Sure™ Histone H3 Peptide Array ELISA Kit (Colorimetric) is a complete set of optimized reagents designed to rapidly examine the selectivity and specificity of antibodies against 46 different histone modifications in a simple, ELISA-like format with use of a standard microplate reader. It is also suitable for identifying substrates of histone modifying enzymes as well as for analyzing specificity of histone binding proteins. The kit has the following advantages and features:
- Quick and efficient 1 hour and 45 minute procedure.
- High quality peptides (over 95% pure) are purified by HPLC and verified with mass spectrometry, and then coated in two concentrations (2 ng and 20 ng), which allows for the screening of both strong and weak reactive antibodies, enzymes, and histone binding proteins.
- The most comprehensive variety of histone H3 modifications, which contain 46 single modifications including the most important methylation (36), acetylation (8) and phosphorylation (2) modifications.
- Detection in ELISA format, which makes the assay easy and convenient.
- Simple, reliable, and consistent assay conditions.
Histone modifications have also been defined as epigenetic modifiers. Post-translational modifications of histones include the acetylation on specific lysine residues by histone acetyltransferases (HATs), the deacetylation by histone deacetylases (HDACs), the methylation of lysine and arginine residues by histone methyltransferases (HMTs), the demethylation of lysine residues by histone demethylases (HDMTs), and the phosphorylation of specific serine groups by histone kinases (HKs). Next to DNA methylation, histone acetylation and histone methylation are the most well-characterized epigenetic marks. Generally, tri-methylation at H3-K4, H3-K36, or H3-K79 results in an open chromatin configuration and is, therefore, characteristic of euchromatin. Euchromatin is also characterized by a high level of histone acetylation, which is mediated by histone acetyltransferases. Conversely, histone deacetylases have the ability to remove this epigenetic mark, which leads to transcriptional repression. Lysine residues can be mono-, di-, or tri-methylated, each of which can differentially regulate chromatin structure and transcription. Along with other histone modifications such as phosphorylation, this enormous variation leads to a myriad of possible combinations of different modifications. This might constitute a “histone code”, which can be read and interpreted by different cellular factors.
Principle & Procedure
In an assay with this kit, the histone H3 proteins modified at specific sites that are tightly arrayed on the wells through biotin-avidin binding are incubated with input materials such as antibodies, proteins or enzymes. After incubation, the specifically bound input materials are detected through an ELISA reaction system (antibody-signal development reagent). The binding intensity of input materials is proportional to the intensity of absorbance.
For antibody screening, the input materials are various IgG antibodies of interest. For histone modifying enzyme/protein screening, purified enzymes/proteins should be used. The amounts or concentrations of input materials are dependent on the assay types and can be determined by users.
Fig. 4. Working principle of Pre-Sure™ Histone H3 Peptide Array ELISA Kit (Colorimetric).
Fig. 1. Schematic procedure of the Pre-Sure™ Histone H3 Peptide Array ELISA Kit (Colorimetric).
Fig. 2. Histone peptide array mapping.
Fig. 3. Representative specificity identification of histone H3 antibodies. The Histone H3 Array was probed with H3K9me1 Polyclonal Antibody (Cat. #A-4034; 1 µg/mL). Peptides and control were visualized using a goat anti-rabbit IgG-HRP and a color development system. A: original image; B: graphical analysis of specificity and binding intensity of H3K9me1 antibody.