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HTRF Human and Mouse Total Histone H3 Detection Kit, 500 Assay Points
The total Histone H3 kit detects the nuclear Histone H3 level and can be used as a normalization assay for the phospho-Histone H3 kit.
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Product information
Overview
The Total Histone H3 cellular assay kit is used as a normalization assay with phospho-Histone H3 kits. Histone H3 is a key regulator of gene expression and mitosis, making it a powerful tool for anti-cancer research.
Specifications
| Assay Points |
500
|
|---|---|
| Assay Target Type |
Kit
|
| Assay Technology |
HTRF
|
| Brand |
HTRF
|
| Quantity |
1
|
| Shipping Conditions |
Shipped in Dry Ice
|
| Therapeutic Area |
Neuroscience
|
| Unit Size |
500 Assay Points
|
Video gallery
How it works
Total-Histone H3 assay principle
The Total-Histone H3 assay quantifies the expression level of Histone H3 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-Histone H3 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of Histone H3 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the proteins expression under a no-wash assay format.
Total-Histone H3 2-plate assay protocol
The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total Histone H3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-Histone H3 1-plate assay protocol
Detection of total Histone H3 with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Assay validation
Total-Histone H3 as normalization control for phospho-Histone H3 (Ser10) assay
NIH3T3 cells (12.5KC per well) were seeded in a 96-well plate and treated with Nocodazole, an antimitotic agent that depolymerizes microtubules and blocks the cells in mitosis, to stimulate Histone H3 (Ser10) phosphorylation. Cells were (experiment a) or weren't (experiment b) co-treated for 16 hours with an inhibitor Danusertib. After cell culture medium removal, 200 µL of supplemented lysis buffer were added for a 30-minute incubation. 10 µL of lysates each were transferred to a 384sv white plate for HTRF analysis in parallel with the total and the phospho-Ser10 assays
Simplified pathway
Total-Histone H3 cell signaling
Histone3 is one of the 5 major canonical Histone proteins to compact DNA into chromatin. The main globular histone domains remain in the long N-terminal Histone tails that are highly post-translationally modified and are responsible for the constitution of either highly condensed heterochromatin or less condensed euchromatin. The different types of coordinated modifications, like acetylation, methylation and phosphorylation, compose the histone code, read by the remodeling machinery. Thus Histone modifications play a major role in the dynamic and long term regulation of genes, making the surrounding DNA more or less accessible to the transcriptional machinery. Phosphorylation of Ser10 or Thr3 in the tails of Histone H3 is involved in gene expression and during mitosis.
Resources
Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
SDS, COAs, Manuals and more
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