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HTRF Human Total H2AX Detection Kit HTRF®

The Total H2AX kit is designed to monitor the expression level of cellular human H2AX, and can be used as a normalization assay for the Phospho-H2AX (Ser139) Detection kit.

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  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption
  • No-wash No-wash
  • High sensitivity High sensitivity

The Total H2AX kit is designed to monitor the expression level of cellular human H2AX, and can be used as a normalization assay for the Phospho-H2AX (Ser139) Detection kit.

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Overview

The Total-H2AX kit offers quantitative detection of human histone variant H2AX. The specific phosphorylation of H2AX at Ser139 may occur when double-stranded DNA breaks, either due to ionizing radiation or to endogenous physiological processes. This modification, called gamma-H2AX (phospho-Ser139), triggers cell cycle arrest and DNA damage response repair, which makes this assay particularly appropriate for studying genome stability, cell cycle, DNA repair, and more broadly as a biomarker for cancer and for aging process investigations.

Benefits

  • SPECIFICITY
  • PRECISION
  • COMPOUND PROFILING

Total H2AX assay principle

The Total-H2AX assay quantifies the expression level of H2AX in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-H2AX 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 H2AX 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 protein’s expression under a no-wash assay format.

Principle of the HTRF total H2AX assay

Total-H2AX 2-plate assay protocol

The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of the Total H2AX HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF total H2AX assay

Total-H2AX 1-plate assay protocol

Detection of Total H2AX 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.

One-plate protocol of the HTRF total H2AX assay

Pharmacological validation with Neocarzinostatin compound

HEK293 cells were cultured in a 96-well plate (12,500 cells/well) for 24 hours at 37°C, 5% CO2. After cell culture medium removal, they were treated with increasing concentrations of Neocazinostatin (DNA damaging agent). After a 30 min incubation, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#4 (1X) were dispensed into each well. After 45 min of cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total H2AX or Phospho-H2AX (Ser139) detection antibodies were added. The HTRF signal was recorded after a 2h incubation.


The Neocazinostatin triggered a dose-dependent increase in Phosphorylated H2AX (Ser139) protein, while the expression level of H2AX remained stable..

Total and phospho H2AX Neocarzinostatin activation

Specificity of HTRF Total H2AX assay using knockout H2AX cell lines

The Human H2AX expression level was assessed with the HTRF Total H2AX kit in HAP1 cells (WT) and HAP1 knocked-out (KO) for H2AX. The cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

The cell lines were cultured in a 96-well plate (50,000 cells/well) for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X) for 45 min, then 16 µL of cell lysate were transferred into a low volume white microplate followed by 4 µL of premixed detection reagents. The HTRF signal was recorded after a 2h incubation at RT.

In HAP1 KO H2AX cells, the HTRF signal was equivalent to the non-specific signal (dotted line), indicating a complete H2AX gene silencing and demonstrating the specificity of the HTRF Total H2AX kit.


HAP1 Ko cell line from Horizon Discovery # HZGHC005630c003

HAP1 Wt from Horizon Discovery # C631
HTRF Total H2AX assay specificity using Ko H2AX

H2AX Simplified pathway

DNA Double-Strand Breaks (DSB) are the most deleterious type of DNA damage, consecutive to endogenous processes or exogenous factors (e.g. ionizing & UV radiations, specific anticancer drugs). H2AX is a variant of the histone H2A family, which becomes phosphorylated at Ser139 upon the event of a DSB. This modification is called gamma-H2AX and within minutes it is spread to thousands of H2AX proteins in proximity to the damage site. It is crucial to activating the DNA damage response pathway, a complex molecular mechanism to detect and repair DNA damage.

Pathway for H2AX

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Product Insert H2AX total Kit / 64H2AXTPEG-64H2AXTPEH

64H2AXTPEG-64H2AXTPEH - Product Insert

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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