作者wei19850210 (we~we)
看板Biotech
标题Re: [方法] NAALADase Assays ?
时间Sun Feb 28 01:57:15 2010
※ 引述《wei19850210 (we~we)》之铭言：
: 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]
就是此酵素活性测定原理不是很了?
对於这方面 算是初学
可否大致说明 使我能较快理解
谢谢!!
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