Detection of Platinum-DNA Adducts

Platinum-DNA adducts of cisplatin-treated cells were detected as previously described (28 (link)). Briefly, cells were placed on Superfrost Plus Gold slides (Thermo Scientific) and air-dried. After fixation in methanol cells underwent a mild alkali-denaturation treatment followed by a two-step proteolytic digestion with pepsin and proteinase K to remove cellular proteins. After blocking, Platinum-DNA adducts were detected using the Pt-[GG]-specific antibody R-C18 (28 (link)) and a Cy3-labeled secondary antibody (Dianova). Nuclei were counterstained by DAPI.

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Bretz A.C., Gittler M.P., Charles J.P., Gremke N., Eckhardt I., Mernberger M., Mandic R., Thomale J., Nist A., Wanzel M, & Stiewe T. (2016). ΔNp63 activates the Fanconi anemia DNA repair pathway and limits the efficacy of cisplatin treatment in squamous cell carcinoma. Nucleic Acids Research, 44(7), 3204-3218.

Publication 2016

Superfrost Plus Gold slides

Adducts Alkali Antibody Cellular Dapi Dna adducts Dna cisplatin adducts Gold Methanol Nuclei Pepsin Platinum Proteinase k Proteins Proteolytic

Corresponding Organization :

Other organizations : Philipps University of Marburg, Universitätsklinikum Gießen und Marburg, University of Duisburg-Essen, Universities of Giessen and Marburg Lung Center, German Center for Lung Research

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Variable analysis

independent variables

Cisplatin treatment

dependent variables

Platinum-DNA adducts

control variables

Cells placed on Superfrost Plus Gold slides
Cells air-dried
Cells fixed in methanol
Mild alkali-denaturation treatment
Two-step proteolytic digestion with pepsin and proteinase K
Blocking
Pt-[GG]-specific antibody R-C18 used for detection
Cy3-labeled secondary antibody used
Nuclei counterstained by DAPI

controls

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

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