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Enzymatic Assay

The tissues were homogenized and suspended in extraction buffer (50 mM Tris-HCl pH 7.6, 2 mM dithiothreitol (DTT), 5 mM benxamidine, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 20% glycerol and 0.5% Nonidet P40). The suspension was centrifuged at 13,000 rpm for 20 minutes and the supernatant collected and stored at -80°C. The protein concentrations were determined using Bradford Protein Assay reagent (Bio-Rad) and bovine serum albumin (BSA) as a standard.

The enzymatic assays were performed in 50mM Tris-HCl (pH 7.6), 5 mM MgCl2, 5 mM ATP, 2 mM DTT, 15 mM NaF, 0.5mg/ml BSA, 40-50µg protein, 3 µM [methyl-3H]thymidine   (Moravek) and 7 µM unlabelled thymidine. 10µL of the reaction mixture was spotted on Whatman DE-81 filter discs after incubation at 37°C at different time points (0, 10, 20 and 30 minutes). The filters were washed three times in 5 mM ammonium formate and the filter bound product was eluted from the filter with 0.1 M KCl and 0.1 M HCl. The radioactivity was quantified by scintillation counting using 3ml scintillation buffer.

Two mice from each group (Dm-dNK positive and wild-type) and for each time point (1 month, 3.5 months and 5 months old) were analyzed. All experiments were performed in triplicates. The data were analyzed statistically (student’s t-test using Prism 5.0 software).


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Last updated: 05/22/11