Western Blot Analysis of Phosphorylated Histone H2AX

Western blot analysis was performed as previously reported [51 (link)]. Briefly, HeLa cells were collected and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 250 mM NaCl, 0.1% Triton) complemented with protease (Thermo Scientific, A32953) and phosphatase inhibitors (Thermo Scientific, 88667). Total proteins were fractionated using SDS-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL, USA). Membranes were probed with the following primary antibodies: mouse mAb anti-phospho-histone H2AX (γH2AX; Millipore, 05-636) and mouse mAb anti-β-actin (Sigma-Aldrich, A5441).

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Platella C., Ghirga F., Musumeci D., Quaglio D., Zizza P., Iachettini S., D’Angelo C., Biroccio A., Botta B., Mori M, & Montesarchio D. (2023). Selective Targeting of Cancer-Related G-Quadruplex Structures by the Natural Compound Dicentrine. International Journal of Molecular Sciences, 24(4), 4070.

Publication 2023

phosphatase inhibitors nitrocellulose membrane anti-phospho-histone H2AX (γH2AX; anti-β-actin

Actin Antibodies Buffer Edta Hela cells Histone h2ax Inhibitors Membranes Mouse Nacl Nitrocellulose Phosphatase Protease Proteins Sds polyacrylamide gel electrophoresis Tris Western blot analysis

Corresponding Organization : Sapienza University of Rome

Other organizations : Istituti di Ricovero e Cura a Carattere Scientifico, University of Siena

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of phospho-histone H2AX (γH2AX) protein
Expression levels of β-actin protein

control variables

HeLa cell line
Lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 250 mM NaCl, 0.1% Triton) with protease and phosphatase inhibitors
SDS-polyacrylamide gel electrophoresis for protein fractionation
Nitrocellulose membrane for protein transfer

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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