U.S. Dept Commerce/NOAA/NMFS/NWFSC/Publications
NOAA-NWFSC Tech Memo-14: 32P-Postlabeling Protocols for Assaying Levels of Hydrophobic DNA Adducts in Fish
Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7
Figure 1
Figure 1. Schematic of the 32P-postlabeling assay procedure
Figure 2a Figure 2b
Figure 2. Autoradiograms of one-dimensional chromatograms used in the preparation of [gamma-32P]ATP. a) An aliquot (~0.1 µL) of the [gamma-32P]ATP synthesis mixture was eluted with 1 M lithium chloride. The autoradiogram indicates a successful synthesis of [gamma-32P]ATP. b) Aliquots of the specific activity determination digest were eluted with 0.24 M ammonium sulfate and 8 mM sodium phosphate, pH 7.4
Figure 3
Figure 3. Profiles of 32P-postlabeled 3'mononucleotides from hepatic tissue from fish showing the chromatographic positions of DNA breakdown products. These breakdown products rarely appear in samples that were immediately frozen in liquid nitrogen and then stored at -80°C. These samples were chromatographed using 1 M sodium phosphate, pH 6.0 for D1, 4.5 M lithium formate, 8.5 M urea, pH 3.5 for D3 and 1.6 M lithium chloride, 0.5 M Tris, 8.5 M urea, pH 8.0 for D4.
a) Profile of postlabeled DNA from fish hepatic tissue that was immediately frozen in liquid nitrogen and then stored at -80°C.
b, c) Profiles of DNA breakdown spots that may be observed in n-butanol enhanced 32P-postlabeled samples.
Figure 4
Figure 4. Representative autoradiograms showing the chromatographic profiles 3'5'-dpNp and -pNp nucleotides after micrococcal nuclease and spleen phosphodiesterase digestion of DNA and RNA samples and subsequent phosphorylation with polynucleotide kinase and [gamma-32P]ATP. Excess [gamma-32P]ATP was degraded by apyrase treatment. Samples were chromatographed on polyethylenimine-cellulose sheets prepared in the laboratory and eluted with 0.24 ammonium sulfate and 8 mM sodium phosphate, pH 7.4
Figure 5
Figure 5. Diagram shwoing the directions of each of the chromatography steps and the cut lines (- - - - - - ). The crosses (+) designate the samples origins.
Figure 6
Figure 6. Influence of solvent systems on the separation of DNA adducts extracted from hepatic tissues of oyster toadfish captured at a creosote contaminated site. Commercial PEI-cellulose sheets were used:
a) D3 - 8.5 M urea, 4.5 M lithium formate, pH 3.5:
D4 - 8.5 M urea, 1.6 M lithium chloride, 0.5 M Tris, pH 8.0
b) D3 - 8.5 M urea, 4.5 M lithium formate, pH 3.5;
D4 - isopropanol/4 N ammonia (1:1).
Figure 7
Figure 7. Diagram showing the PEI-cellulose setup for optional D5. The dashed lines show where the chromatogram is to be trimmed after D5 and before being placed in the autoradiography cassette.
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