如题~ 我有点搞不清楚这段文章的叙述 对於这技术方法很不了解 不知道有没有熟悉的好心人指导指导? 文章内容如下: NAALADase Assays. NAAG hydrolysis was performed essentially as described previously (9). In short, LNCaP cell extracts were prepared by sonication in NAALADase buffer [50 mM Tris (pH 7.4) and 0.5% Triton X-100]. Using this buffer, either cell lysate or purified xPSM was incubated in the presence of the radiolabeled substrate N-acetyl-L-aspartyl-L-(3,4-3H)glutamate (NEN Life Science Products, Boston, MA) at 37°C for 10–15 min. The reaction was stopped by the addition of an equal volume of ice-cold 100-mM sodium phosphate and 2 mM EDTA. Products were partitioned by AG 1-X8 formate resin (Bio-Rad Laboratories) anion exchange chromatography, eluted with 1 M sodium formate, and quantitated by scintillation counting. In general, aptamer IC50 was determined in the presence of 8 nM NAAG with serially diluted RNA. Aptamer Ki was determined using 5–30 nM aptamer in the presence of serially diluted substrate. General NAALADase assays and aptamer inhibition experiments showed no significant differences when done in the presence of 1 mM of cobalt chloride. Trend lines were determined using linear regression for Ki plots and logarithmic trend lines for enzyme kinetics, applying R2 to determine fitness. Points > 3 SD away from the mean were considered outliers. Competitive inhibition Ki s were calculated by changes in Km, where noncompetitive inhibition Kis were calculated by changes in Vmax. Aptamer inhibition experiments were repeated at least once to confirm affinity and mode of inhibition. Experiments were designed to allow 20% or less of the total substrate to be cleaved. By~ [CANCER RESEARCH 62, 4029–4033, July 15, 2002] --

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