실험 Q&A Chemical biology > Analysis
연구를 이제 시작해보는 초짜 대학원생입니다.(Neprilysin 관련)
레벨1 사용가능한닉네임 (과기인)
안녕하세요. 이제 연구를 처음 시작하는 대학원생이라 모르는 게 많아
도움좀 구하고자 글 남깁니다.
Ayoub. S. Natural Product Communications. 2006. 1. 633
Effects of flavonoids on NEP and ACE ~~ 논문에서
Bormann and Melzig method 라면서 블라블라 나오는데요.
Neprilysin activity 측정하는 방법 중에 하나인듯 해요.
혹시 이 실험 과정에 대해 알고 계시는 분 있으시면 조언 부탁드립니다.
아래는 간략히 정리한다고 하면서 써있는 글입니다.
(제 글이 혹시 표절? 등의 문제가 있다고 한다면 말씀해주세요. 지우겠습니다. 다른 따끔한 조언 등도 달게 받겠습니다.)
Briefly, 20 μL of Hip-LHis-
L-Leu solution (24 mM in water) and 260 μL of
phosphate-buffer (83 mM K2HPO4 X 3 H2O + 326
mM NaCl, pH 8,3) were added to the intact cell layer
after removing the growth medium. To investigate
the acute inhibitory activity of a test substance this
compound was simultaneously added. The cells with
assay solution were incubated for 20 min at 37°C.
The incubation mixture of each well (250 μL) was
transferred to an Eppendorf tube and the reaction was
stopped by the addition of 1 mL 0.4 N NaOH, plus
100 μL o-phthalaldehyde solution (2 % in methanol,
freshly prepared). Under exclusion of light this
mixture was incubated for 10 min at room
temperature. Adding 300 μL 2 N HCl terminated the
reaction. The fluorescence of the formed product was
measured at λexit =360 nm, λemiss =500 nm and slit
=3nm. To calculate the enzyme activity, a calibration
curve with L-His-L-Leu was determined. During the
enzymatic reaction and considering the absorbance of
fluorescent light by test compounds, the inhibition
rates were calculated in comparison to controls
without an inhibitor. Enzyme activity was calculated
in pmol/min per 100,000 cells for ACE and NEP. In
all investigations the effect of DMSO was considered
in separate control experiments.
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