32p atp

The aptamer–protein equilibrium dissociation constants (Kd) were determined via the nitrocellulose-filter binding method (32 (link)). For all binding assays, aptamers were dephosphorylated using alkaline phosphatase, 5-end labeled using T4 polynucleotide kinase (New England Biolab) and [³²P]-ATP (Amersham Pharmacia Biotech). Direct binding assays were carried out by incubating a ³²P-labeled aptamer at a concentration of <10 pM and protein at concentrations ranging from 10 pM to 100 nM in a selection buffer. The fraction of bound aptamer was quantified with a PhosphorImager (Fuji FLA-5,100 Image Analyzer). Raw binding data were corrected for non-specific background binding of radiolabeled aptamer to the nitrocellulose filter.

All subjects were asked not to eat fried, grilled, or barbecued meat during the 24 h before sampling.
Chemicals: Tritiated (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10 tetra-hydro-benzo[a]pyrene (3H-anti-B[a]PDE), with a specific activity of 1,941 mCi/mmol, was obtained from the NCI Chemical Carcinogen Standard Repository (Bethesda, MD, USA). The purity and authenticity of the B[a]P metabolite were determined by the NCI Carcinogen Standard Repository. [32P] ATP (>5000 Ci/mmol) was obtained from Amersham (UK). All other reagents were of the purest grade available. Sample Collections: Peripheral venous blood samples were collected from coke oven workers in heparinized vacutainer tubes after at least three consecutive days of PAH exposure and maintained in refrigeration during transport. The PBLs were isolated on Ficoll separating solution (Seromed, Germany), as previously described [56 (link)] and frozen at −20 °C for later DNA isolation.
DNA Isolation: DNA from human PBLs was isolated following the original procedure described by Johns [57 (link)] with minor modifications [58 (link)]. PAH and B[a]P lymphocyte DNA adducts with p32 post-labelling. DNA adducts were detected essentially as described by Reddy [59 (link)], with minor modifications [60 (link)]. All determinations were carried out in duplicate [61 ].

Total RNA was isolated from different tissues from adult chickens and chicken embryos using TRIzol (Invitrogen, Carlsbad, CA, USA). Total RNA (20 µg) was fractionated using a 15% denaturing polyacrylamide/8M urea gel. The RNA was then transferred to a GeneScreen Plus membrane (Perkin–Elmer, Waltham, Massachusetts, USA). The 5′ ends of the DNA probes were labeled with [³²P]ATP (Amersham, Pittsburgh, PA, USA) using T4 polynucleotide kinase (New England BioLabs, Ipswich, Massachusetts, USA). Hybridization and washing were performed as described [24] (link). Hybridization signals were detected using a Phosphor Screen (Molecular Dynamics, Sunnyvale, CA, USA).