Determination of DNA Lesions Monoclonal Antibody Method

Plant material was collected simultaneously with the gel-electrophoresed samples (samples collected in July 1999) and stored at - 80°C until processed. Samples were shipped on dry ice to the University of Tennessee at Chattanooga, Chattanooga, TN for processing. As described in A. E. Stapleton (1999) and the references therein, DNA was isolated from leaf tissue powdered in liquid nitrogen. Samples were processed by adding ~500 ng of DNA to TE buffer, followed by denaturation and slot-blotting to the membrane. CPDs were detected using a TDM-2 monoclonal antibody. This method detects the primary-bound antibody by an alkaline phosphatase-conjugated secondary antibody. By using a chemiluminescent substrate (CSPD), chemiluminescence was detected with autoradiographic film (or with a cooled CCD camera), and the counts were plotted as a function of time.

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