[方法] NAALADase Assays ?
如題~
我有點搞不清楚這段文章的敘述
對於這技術方法很不了解 不知道有沒有熟悉的好心人指導指導?
文章內容如下:
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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