• Biomolecules & Therapeutics
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• 제18권2호
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• pp.152-158
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• 2010

Melanin concentrating hormone is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G-protein coupled receptor family, especially plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome. Although fluorescence-based calcium mobilization assay platform has been one of the most widely accepted tools for receptor research and drug discovery, fluorescence interference and shallow assay window limit their application in high throughput screening and have led to a growing interest in alternative, luminescence-based technologies. Herein, a luminescence-based functional assay system for the MCH1 receptor was developed and validated with the mitochondrial targeted aequorin. Aequorin based functional assay system for MCH1 presented excellent Z' factor (0.8983) and high signal-to-noise ratio (141.9). The nonpeptide MCH1 receptor antagonist, SNAP 7941 and GSK 803430, exhibited $IC_{50}$ values of 0.62 ${\pm}$ 0.11 and 12.29 ${\pm}$ 2.31 nM with excellent correlation coefficient. These results suggest that the aequorin based assay system for MCH1 is a strong alternative to the traditional GPCR related tools such as radioligand binding experiments and fluorescence functional determinations for the compound screening and receptor research.

• 생명과학회지
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• 제18권8호
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• pp.1159-1163
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• 2008

이 연구의 목적은 물-오일 계면에서 특이적인 lipase 활성을 측정할 수 있는 high-throughput assay 방법을 확립하는 것이다. 이 방법은 pH에 따라 형광의 세기가 변하는 fluorescein의 특성을 이용하여 lipase의 작용으로 방출되는 지방산으로 인한 pH 변화를 fluorescein의 형광 변화로 측정하도록 되어 있다. 활성의 측정은 오일 에멀션과 fluorescein 그리고 효소를 포함하는 반응 용액을 반응시키면서 일정한 간격으로 형광을 측정함으로써 이루어진다. 이 방법을 통해 형광의 세기가 효소의 양에 비례하는 속도로 감소하는 것을 관찰할 수 있었으며 시간에 따른 형광 변화 그래프로부터 계산한 반응 속도가 효소의 양에 선형으로 비례한다는 것을 확인할 수 있었다. 또 한 가지 중요한 사실은 assay를 하는데 있어서 pH 6.0-8.0의 범위에서 다른 pH 조건을 사용할 수 있었다는 점이다.

• BMB Reports
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• 제36권4호
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• pp.421-425
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• 2003

The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phosphothreonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the $K_m$ for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.

• 센서학회지
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• 제14권5호
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• pp.308-312
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• 2005

We present a microfluidic chip designed for the detection of antibody by using quantum dots fluorescence and a microbead-based assay. A custom designed PDMS microfluidic chip with multi-layer channel is utilized for capturing microbeads; antibody injection into each micro-well; QD injection; and fluorescence detection. The experiment using the fabricated microfluidic chip has been performed on solutions with various concentrations of antibody and has shown correlated fluorescent intensities.

• BMB Reports
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• 제30권4호
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• pp.258-261
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• 1997

A simple quantitative assay method for ribonuclease activity has been developed. This method is based on the decrease of fluorescence intensity emitted by the ethidium bromide bound to RNA due to the degradation of RNA by ribonuclease. The substrate RNA was reacted with ribonuclease A and the fluorescence intensity was measured after the addition of ethidium bromide. The intensity difference was calculated using a blank reaction mixture containing no RNase. Whole cellular RNA substrate produced a significant error and was not suitable for this assay method possibly because of local microheterogeniety caused by high molecular weight rRNA. but satisfying results were obtained with tRNA substrate. The intensity difference increased linearly by raising enzyme concentration up to $2{\times}10^{-4}$ Kunitz Units of ribonuclease A. More refined and reliable results were obtained by use of initial reaction velocities which were calculated from the plots of intensity difference vs time. A linear relationship between initial velocities and enzyme concentrations was observed up to 0.01 Kunitz Units of enzyme.

• 대한의생명과학회지
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• 제16권4호
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• pp.279-285
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• 2010

Endogenous cannabinoids (endocannabinoids) display various pharmacological effects including pain control, anti-inflammation, and neuroprotection. The synthesis and release of endocannabinoids are regulated under both physiological and pathological conditions. The main degrading enzyme of endocannabinoid is fatty acid amide hydrolase (FAAH). Therefore we have developed the fluorescence-based assay system for FAAH. We established stable CosM6 cell lines expressing human FAAH. We also synthesized 2-oxo-2H-chromen-7-yl decanoate (DAEC) as a fluorogenic substrate for FAAH. When crude membrane extracts stably expressing FAAH was incubated with DAEC at $25^{\circ}C$, FAAH reacted specifically to DAEC and catalyzes the hydrolysis of DAEC into decanoic acid and highly fluorescent coumarin. Furthermore, the serin hydrolase inhibitor, phenylmethanesulfonylfluoride, inhibited the coumarin release to the reaction buffer in concentration dependent manner. This assay system is suitable for high-throughput screening since this system has simple experimental procedure and measurement method.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.169.1-169.1
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• 2003

Recently development of cell-based assay systems which are useful in molecular cell biology and drug discovery attracts significant attention. Here, we introduce a new technologies for monitoring enzyme activity and its inhibition inside living cells. Among various enzymes, proteases are important targets for studying various biological and disease-related processes such as viral infections, apoptosis and Alzheimer's disease. In this study, a sensitive cell-based protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells is introduced. (omitted)

• Weed & Turfgrass Science
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• 제4권4호
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• pp.308-314
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• 2015

본 연구는 식물세포막을 파괴시켜 제초활성을 나타내게 하는 화합물(막과산화형 제초제)을 미지의 많은 화합물로부터 신속하게 탐색하기 위한 새로운 검정법을 확립하기 위하여 실시되었으며, 확립한 전체적인 검정과정은 다음과 같다. 96-well microplate에 시험용액 $200{\mu}L$ 넣고 여기에 오이자엽으로부터 적출한 직경 4 mm의 절편 1개씩을 띄운다. 항온실의 광조건 하에서 회전진탕기로 조금씩 흔들어주면서 8시간 배양한 후 절편을 제거한 다음, 배양액에 HVA와 HRP를 첨가하여 반응시킨 후 마이크로평판용 형광검출기를 이용하여 형광도(Ex 320 nm, Em 425 nm)를 측정한다. 형광변화량이 높을수록 제초활성이 높은 것으로 판단한다. 본 방법은 96-well microplate에서 작업을 수행할 수 있고 형광검출 기술을 이용함으로써 검정과정과 작업을 간편하게 하여 검정효율을 기존보다 현저히 높인 것이 특징이다. 아울러 활성검정을 추출된 효소가 아닌 잎 절편수준에서 수행하기 때문에 보다 실용화에 근접한 정량적 데이터를 얻을 수 있는 장점을 가진다.

• 대한화학회지
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• 제65권5호
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• pp.397-400
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• 2021

• KSBB Journal
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• 제16권6호
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• pp.609-613
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• 2001

형광을 이용하여 중금속 이온의 농도를 측정할 수 있는 신규 단백질 바이오센서를 제작하였다. 바이오센서의 transducer로 카제인과 알부민을 사용하였을 때 $10^{-3}$mM-1 mM 범위에서 중금속 농도 정량에 우수성을 보였다. 또한 두 가지 중금속 혼합물에서 excitation과 emission파장에서 형광도를 측정하여 각 중금속 이온 농도를 선택적으로 정량하는 방법을 제시하였고, Co$^{2+}$, Fe$^{3+}$ 이온 혼합물을 대상으로 본 논문에서 제시한 방법의 유효성을 입증하였다.