Western Blot Analysis of DNA Damage Response

Whole cell extracts for western blotting were prepared as described previously (17 (link)). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto Immobilon-FL PVDF membranes (Millipore) according to standard procedures. Membranes were probed with the following antibodies: APE1 (Novus Biologicals, NB100–101), XRCC1 (Thermo Scientific, MS-1393-P0), α-actin (Abcam, ab6276), p21 (Cell Signaling Technology, 2947), p53 (Santa Cruz, sc-126), PAR (Trevigen, 4335-AMC-050), histone H2A.X phosphorylated at Ser139 (γH2AX, 05–636, Millipore), Sp1 (Millipore, 07–645), histone H3 (Cell Signaling Technology, 4499). Secondary antibodies conjugated with Alexa Fluor 680 (Molecular Probes) and IRDye^® 800 (Rockland) fluorescent dyes were used. Detection and quantification was carried out using an Odyssey image analysis system (Li-Cor Biosciences).

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Poletto M., Legrand A.J., Fletcher S.C, & Dianov G.L. (2016). p53 coordinates base excision repair to prevent genomic instability. Nucleic Acids Research, 44(7), 3165-3175.

Publication 2016

Immobilon-FL PVDF membranes XRCC1 α-actin histone H2A. γH2AX histone H3 Alexa Fluor 680 IRDye® 800 Odyssey image analysis system

Actin Antibodies Ape1 Biologicals Cell Fluorescent dyes Histone h2a x Histone h3 Immobilon Irdye 800 Membranes Molecular probes Novus Proteins Pvdf Sodium dodecyl sulphate polyacrylamide gel electrophoresis Xrcc1

Corresponding Organization :

Other organizations : CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Institute of Cytology and Genetics

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of APE1, XRCC1, α-actin, p21, p53, PAR, γH2AX, Sp1, histone H3

control variables

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation
Transfer of proteins onto Immobilon-FL PVDF membranes
Probing of membranes with specific antibodies
Use of secondary antibodies conjugated with fluorescent dyes
Detection and quantification using an Odyssey image analysis system

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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