Anti-gamma H2A.X (phospho S139) antibody - ChIP Grade (ab2893)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Add protease inhibitors to all lysis solutions before use.	-Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Publication protocol

AsiSI-ER-U2OS cells (12) were treated with 300 nM 4-OHT (Sigma) for 4 h to induce AsiSI-ER nuclear localization and DSB generation by the AsiSI nuclease. FokI-U2OS reporter cells (13) expressing inducible ER-mCherry-LacI-FokI-DD were treated with 300 nM 4-OHT plus 1 μM Shield-I (Clontech) for 4 h to induce nuclear expression and stabilization of ER-mCherry-LacI-FokI-DD and DSB generation at the transgene-harboring Lac operator sequences by the FokI nuclease. Subsequently, ChIP was carried out using the EZ-ChIP Kit according to the manufacturer’s protocols (Millipore). The immunoprecipitated and input DNA were analyzed by quantitative PCR (qPCR) using the Rotor-Gene SYBR® Green PCR kit (Qiagen) on a Rotor-Gene Q system (Qiagen). The PCR conditions were an initial preincubation step at 95°C for 5 min followed by 45 cycles of 95°C for 5 s and 60°C for 30 s. The last amplification cycle was followed by a melting curve analysis to confirm the specificity of the PCR amplification. Relative IP values were calculated based on the threshold cycle (Ct) value using the 2−ΔΔCt method (16).

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Manufacturer protocol

Download the product protocol from Abcam for Anti-gamma H2A.X (phospho S139) antibody - ChIP Grade (ab2893) below.

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