The Guava(R) DNA Damage Histone H2A.X Dual Detection Kit ー25 Tests (Part Number: FCCS025153) is part of a series of flow cytometry products that include a pair of antibodies which bind to the same protein; one antibody detects total protein expression and the other detects the phosphorylated form of the same target. Two-parameter analysis allows for the specific detection of phosphorylation and eliminates false positives, enhancing the signal-to-noise ratio.
The Guava DNA Damage Histone H2A.X Dual Detection Kit includes two directly conjugated antibodies?a phospho-specific anti-phospho-Histone H2A.X (Ser139)-PerCP and an anti-Histone H2A.X-FITC conjugated antibody?to measure total levels of Histone H2A.X expression. This two-color kit is designed to detect the extent of Histone H2A.X pathway activation by measuring H2A.X phosphorylation relative to the total H2A.X expression in any given cell population. By doing this, the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of H2A.X activation after treatment. Simultaneous measurement of both total and phospho-Histone H2A.X confirms target specificity of the phosphorylation event.
The antibody pair provided in this kit has been carefully titrated to ensure its ability to measure total and phospho-Histone H2A.X simultaneously for accurate determination of protein level and activation. This kit is a sensitive and valuable tool for studying factors that induce DNA damage and affect DNA repair, allowing exploration of the linkage between DNA damage, cell cycle checkpoints, and initiation of apoptosis.
H2A.X is a member of the Histone H2A family. Histone H2A.X resides downstream of the DNA damage kinase signaling cascade. Phosphorylation of Histone H2A.X at serine 139 is an important indicator of DNA damage. As the level of DNA damage increases, the level of phospho-Histone H2A.X (also known as γH2AX) increases, accumulating at the sites of DNA damage. This accumulation of phospho-Histone H2A.X is often used to indicate the level of DNA damage present within the cell. H2AX is also responsible for recruiting response proteins to the site of DNA damage, and may play a role in DNA repair.
In order to validate that the level of H2A.X activation is accurate, a total H2A.X antibody is multiplexed by flow cytometry to allow for normalization of the total and phospho H2A.X levels, since DNA content levels vary based on which stage cells are within the cell cycle. Histone doubles in content during the cell cycle at the same rate that DNA content doubles. Measuring total H2A.X will account for this increase, so the phosphorylated levels can be accurately determined.
A comprehensive understanding of both Histone H2A.X activity and the consequence of its activation can provide researchers with useful information in characterizing the DNA damage response. The Histone H2A.X DNA Damage Dual Detection Kit is designed to allow the researcher to monitor and accurately measure phospho-specific Histone H2A.X activation in a population of cells.
Components:20X Anti-phospho-Histone H2A.X (Ser139)-PerCP, 20X Anti-Histone H2A.X-FITC, Fixation Buffer, 10X Wash Buffer, 5X Assay Buffer, 1X Permeabilization Buffer
For Research Use Only. Not for use in diagnostic procedures.