The authors thank the Nikon Imaging Center at Istituto Italiano di Tecnologia, Genova, Italy (NIC@IIT).

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The authors have nothing to disclose.

효소 활성은 미세 RNA 전사, 단백질 합성, 단백질 전좌, 번역 후 변형 및 단백질 - 단백질 상호 작용을 포함하여 다양한 수준에서 조절된다. 종종, 효소의 발현 혼자의 활동을 설명하지 않습니다. ABPP는 천연 상태의 단백질의 활성을 연구하기 위해 개발되었다. 두 가지 기능이 요구된다 : 공유 관심 효소 리포터 태그의 활성 부위에 결합하는 화학 프로브는 프로브 - 표지 효소를 검출. 프로브 설계 및 합성 과정의 중요한 포인트입니다. 프로브는 대상에 대한 충분한 친화력 및 선택성이 있어야합니다. 또한, 기자 태그의 존재는 참여 대상에 영향을주지해야합니다. 이 문제는 주로 대상이 잡힌 후에 리포터 태그를 도입 한 두 단계 ABP의 디자인에 의해 극복된다. 태그없는 프로브, 생체 내 연구에 특히 적합하다하는 단백질 활성최소한의 변경으로 외부 살아있는 세포 또는 유기체에 평가 될 수있다. NAAA 프로브 ARN14686은 위에서 설명한 요구 사항을 충족하도록 설계되었습니다. β - 락탐는 반응 탄두는 NAAA 억제제 16,26의 β - 락탐 클래스를 얻을 전에 결과를 바탕으로 선정되었다. 이들 화합물은 공유 결합 된 효소의 촉매 시스테인 결합하여 강력하고 선택하게 NAAA을 억제한다. 또한, 화합물 (16) 전신 활성이있는 것으로 나타났다. 우리는 긴 지방족 사슬에 대한 계정에 NAAA의 증가 친 화성을 복용하는 C9 포화 지방족 사슬을 소개했다. 단말 알킨은 두 단계로 표시 가능하도록 하였다. 다른 중요한 단계는 도우와 생체 내 투여를위한 시점을 선택한다. 이 플라즈마에 프로브, 목표 선호도, 그 선택의 안정성에 따라 달라집니다. 정확한 용량은 포의 참여를 피하면서 목표 캡처 수 있도록 선택해야합니다ssible 오프 대상. 우리는 / kg ARN14686 정맥 3 mg을 선택적으로 NAAA를 캡처 할 최적의 것을 발견했다. 높은 용량은 상동 시스테인 아미 다제, 산 세라 미다의 캡처 결과. 처리 길이에 대해서는, 폐 때처럼 짧은 시간 (2 시간)을 타겟과 반응하는 프로브를 허용하기에 충분할 수있다 잘 관류 기관 분석. 발 들어, 그러나, 반응 시간을 두 배로해야만 하였다. 단백질 얼룩으로 대상 분석 가능한 문제는 자연스럽게 바이오틴 단백질의 존재 때문이다. 이들은 필연적으로 특정 검색 대상과 함께 식별됩니다. 우리는 CC를 수행하기 전에 스트렙 타비 딘의 구슬 preclearing 단계를 도입하는 것은 큰 결과의 품질을 향상 것을 발견했다. 한편, 기본 바이오틴 단백질의 존재는 가능한로드 아티팩트를 통제하는 데 사용될 수있다. 마지막으로, 형광 현미경으로 위치 파악 과정에 관해서, P 인식하는 것이 매우 중요단백질 말 달리 형광 현미경 오프 타겟으로부터 타겟의 차이를 허용하지 않기 때문에 선택성 로브. 예비 선택성 연구 가능성을 평가하고 최적의 실험 조건을 설정하기 위해 수행해야합니다. 상술 한 기술의 한계는 주로 세제 및 아민 - 함유 버퍼를 사용하는 것과 CC 반응에 영향을 미칠 수있는 실험 조건을 회피 할 필요성에 관계. 이러한 측면은 세포 용 해물 또는 조직 균질를 준비 할 때 계정에주의해야한다. 단백질 함유량이 250 μg의 없을 때이 과정에만 적용될 수 있기 때문에 또한, 스트렙 농축 단계가 요구되는 경우, 출발 물질의 양이 다른 문제를 구성한다. 이러한 제한으로 인해 이러한 작업 CC 유도 침전 후 볼륨 단백질 복구 및 스트렙 사용될 수지의 양 기술적 문제로 설정된다. PreviouslY는 활성 NAAA는 효소의 생체 외 활성화 및 가용화 기판 (14)의 기동 버퍼의 사용을 요구 활성 분석을 수행함으로써 평가 될 수있다. 이 방법은 활성화되지 NAAA의 존재에 대해, 총 NAAA 발현에 대한 정보를 제공합니다. 또 다른 가능성은 PEA 및 OEA의 조직 수준을 측정하는 것이지만,이 방법은 34, 35 NAAA 활성을 평가하기 만 간접적 방법을 나타낸다. 또한, FAE 수준은 그러한 생합성 같은 다른 요인에 의해 영향을받을 수있다. 화학 프로브 ARN14686는 NAAA위한 제 ABP이다. 여기에 설명 된 프로토콜이 in vitro 및 in vivo의 쌍방에, 캡처 NAAA의 활성 형태를 가시화하기위한 간단한 방법을 나타낸다. 모든 샘플 조작 따라서 효소의 생체 상태에 대한 신뢰성있는 정보를 제공하는, 프로브의 타겟 반응에 후속한다. 또한, 형광 현미경 ARN14686의 사용은 고유 한 공구를 나타내고 tO를 활성화 NAAA 현지화. NAAA 촉매 서브 유닛을 인식 가능한 항체가의 NAAA 전체 길이 proenzyme 활성 효소를 구별하지 않습니다. 여기에 설명 된 프로토콜부터는 NAAA 발현 및 활성화는 상이한 세포주 및 염증의 동물 모델에서 분석 될 수있다. 공동 지역화 연구가 더 생리적 및 병리 적 조건에서 NAAA의 역할을 특성화하기 위해 수행 될 수있다.

일반적 산탄 분석 1,2- 효모 용 액체 크로마토 그래피 질량 분석 플랫폼을 포함하는 발현 패턴의 상호 작용 및 기능 단백질을 조사하는 방법을 사용하는 두 개의 하이브리드 방법 3,4 및 시험 관내 분석에서 그들이 것을 한정 그 나라의 상태에서 단백질의 활성을 평가할 수 없습니다. 활동 기반 단백질 프로파일 (ABPP)이 갭을 채우기 위해 사용될 수있다. 이 방식에서, 작은 분자에 공유 표적 탐지 가능 리포터 그룹에 결합되는 관심있는 효소의 활성 부위에 결합 할 수있는 프로브. 클릭 화학 (CC)를 사용하여 리포터 프로브에 통합 될 수 있거나 또는 표적 결합이 5,6 발생한 후에 도입 될 수있다. 후자의 방법은 SUC 바이오 직교 반응을 통해 리포터 시약 다수 개질 될 수있는 단자 알킨 또는 아 지드와 같은 적합한 화학 그룹을 포함하는 프로브를 사용해야구리 (I)와 같은 시간 [3 + 2] 사이클로 7-9 또는 슈타 우 딩거 결찰 10, 11 Huisgen을 촉매 반응. 최근에는 체외위한 제 ABP와 같은 시스테인 가수 분해 효소, NAAA (12)의 생체 검출의 화합물 ARN14686 개시된. NAAA는 항 염증 핵 수용체 PPAR 알파 13 ~ 15의 내인성 작용제이다 oleoylethanolamide (OEA) 및 팔미 토일 (PEA)을 포함하여 포화 및 불포화 FAES,의 가수 분해 비활성화를 촉매한다. NAAA 주로 선천성 면역 반응의 조절에 중요한 역할을 시사뿐만 B 림프구 (14,16)와 같이, 대 식세포 및 다른 단핵구 유래 된 세포에서 발현된다. 효소는 비활성 형태로 거친 소포체에서 합성 및 autoproteolytic기구 (17)에 의해 세포의 산성 구획에 활성화됩니다. autoproteolytic 분열은 (C13를 새 N 말단 시스테인을 생성마우스 및 쥐에서 1, 인간의 C126), 즉 FAE의 친핵체 책임은 (18, 19)을 가수 분해한다. NAAA 활동의 약리 억제는 FAES 16,20,21 증가 세포 수준에 찬성 FAE 합성 / 분해의 균형을 변경합니다. 여러 β-락톤 및 β - 락탐 유도체는 높은 효능 및 선택성 16,22-26와 NAAA 활성을 억제하는 것으로 나타났다. 이러한 억제제는 촉매 시스테인 16,27,28의 S의 -acylation을 통해 작용한다. (- [(S) -2- oxoazetidin -3- 일] 카르 바 메이트 4- cyclohexylbutyl- N) 16 화합물 ARN14686은 전신적 활성 세린 유래 β 락탐 NAAA 억제제의 화학 구조, ARN726에 기초하여 설계되었다. ARN726의 4 부틸 사이클로 헥실 그룹은 아 지드 베어링 기자 태그 이후 CC의 접합을위한 터미널 알킨 태그를 베어링 C9 포화 지방족 체인으로 대체되었다. 우리 minimall하는 두 단계 ABP 디자인을 선택따라서 Y NAAA 용 프로브의 친화력을 유지하면서 원래의 지지체의 구조를 변화. 또한, 부피가 태그의 도입을 피하는 등의 프로브가 직접 ABP보다 생체 내 치료에 적합 할 수있다. ARN14686 높은 효능으로 NAAA 억제 효소 촉매 (12)의 시스테인과 공유 부가 물을 형성함으로써 (hNAAA IC 50 = 6 nM의, rNAAA IC 50 = 13 ㎚). 살아있는 쥐의 실험 프로브가 NAAA가 폐에 표현 캡처 선택적 있음을 보여 주었다. 높은 프로브 농도 (시험 관내에서 10 μM, 10 밀리그램 / 정맥 주사, 정맥 ㎖) (12)를 사용할 때 산성 세라 미다, NAAA와 33~34% ID를 공유하는 다른 시스테인 아미 다제는 또한 낮은 친 화성 대상으로 확인되었다. 우리는 또한 완전한 프로 인트 보조제 (CFA) (29)의 투여 후 염증이 쥐의 조직에서 활동 NAAA의 존재를 연구하는 ARN14686을 사용했다. 여기에서는 preparati위한 프로토콜 개요ARN14686 (그림 1)과 NAAA 활성화 생체의 조사에의 응용의에. 예를 들어, 우리는 CFA 투여 후 쥐의 발에 NAAA을 시각화하는 실험 절차를 설명합니다. 이 실험에서, 단백질은 프로브의 IV 주입 후 발 조직으로부터 추출되고, ABP 표지 프로테옴 비오틴 지드와 CC 받는다. 비오틴 샘플을 스트렙 타비 딘 비드를 사용하여 농축하고, 단백질 블롯 행한다. 다른 응용 프로그램에서, 우리는 프로브 처리 된 마우스에서 마우스의 폐에서 형광 현미경으로 활성화 NAAA의 현지화를 설명합니다. 이 경우, 조직을 절단하고, 절편을 로다 민 첨가 CC 실시된다. 워크 플로 방식은도 2에 도시되어있다.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">1,1&#8217;-sulfonyldiimidazole&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">367818</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">2-dipyridylcarbonate</td>
			<td style="width:205px;">Fluorochem</td>
			<td style="width:111px;">11331</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">2-Methylbutan</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">M32631</td>
			<td>Flamable, toxic,hazardous to the aquatic environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">4-(Dimethylamino)pyridine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">107700</td>
			<td style="width:471px;">Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Acetic acid</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">695092</td>
			<td style="width:471px;">Flammable, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Acetonitrile&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34998</td>
			<td style="width:471px;">Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Activated charcoal</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">161551</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ammonium chloride</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A9434</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azide-PEG3-Biotin</td>
			<td style="width:205px;">Jena Biosciences</td>
			<td style="width:111px;">CLK-AZ104P4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azide-PEG3-Fluor 545</td>
			<td style="width:205px;">Jena Biosciences</td>
			<td style="width:111px;">CLK-AZ109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">BCA protein assay kit</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bio-spin columns</td>
			<td style="width:205px;">Biorad</td>
			<td style="width:111px;">732-6204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Biotin</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Blocking buffer</td>
			<td style="width:205px;">Li-Cor Biosciences</td>
			<td style="width:111px;">927-40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">b-mercaptoethanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">M6250</td>
			<td>Higly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bovin serum albumine (BSA)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bromophenol blue</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">B0126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bruker Avance III 400</td>
			<td style="width:205px;">Bruker</td>
			<td style="width:111px;"></td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Celite</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">419931</td>
			<td style="width:471px;">Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ceric ammonium nitrate&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">22249</td>
			<td style="width:471px;">Oxidizing, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Chloral hydrate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">C8383</td>
			<td>Higly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">CuSO4.5H2O&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">209198</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cyclohexadiene</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">125415</td>
			<td style="width:471px;">Flammable, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cyclohexane</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34855</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dichloromethane</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34856</td>
			<td style="width:471px;">Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Diethyl ether</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">296082</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:205px;">Acros Organics</td>
			<td style="width:111px;">348441000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethyl sulfoxide d6 (DMSO-d6)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">175943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">2860</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethyl acetate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34858</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Glycerol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Irdye 680-LT Streptavidin</td>
			<td style="width:205px;">Li-Cor Biosciences</td>
			<td style="width:111px;">925-68031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">IRDye680-LT Streptavidin&#160;</td>
			<td style="width:205px;">Licor</td>
			<td style="width:111px;">925-68031</td>
			<td>Briefly centrifuge before use to precipitate protein complexes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Methanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34966</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Methanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34860</td>
			<td style="width:471px;">Flammable, Toxic, Health hazard</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">E7750</td>
			<td style="width:471px;">Harmful, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N,N-diisopropylethylamine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">D125806</td>
			<td style="width:471px;">Flammable, Corrosive, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N,N-dimethylformamide</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">227056</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N-Cbz-L-Serine</td>
			<td style="width:205px;">Fluorochem&#160;</td>
			<td style="width:111px;">M03053</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Nikon A1 confocal microscopy</td>
			<td style="width:205px;">Nikon</td>
			<td style="width:111px;"></td>
			<td>Read&#160; the user manual</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">NuPAGE 4-12% Bis-Tris gel</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">NP0335BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Palladium on carbon</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">330108</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">p-anisidine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A88255</td>
			<td style="width:471px;">Toxic, Health hazard, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Paraformaldehyde</td>
			<td style="width:205px;">sigma Aldrich</td>
			<td style="width:111px;">441244</td>
			<td>Toxic, respiratory harmful, corrosive, falmable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol)&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P3265</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">ProLong Gold antifade mountant with DAPI&#160;</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">P36931</td>
			<td>Avoid bubbles formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Protease inhibitor cocktail</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium bicarbonate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">S6014</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium dodecyl sulfate (SDS)&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">L3771</td>
			<td>Toxic, corrosive, falmmable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium hydride&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">452912</td>
			<td style="width:471px;">Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium sulfate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">239313</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Starion FLA-9000 immage scanner</td>
			<td style="width:205px;">FUJIFILM</td>
			<td style="width:111px;"></td>
			<td>Read&#160; the user manual</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Streptavidin agarose</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td>20349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sucrose</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">S7903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tert-butanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">360538</td>
			<td>Toxic, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tetrahydrofuran</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">186562</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Thiourea</td>
			<td style="width:205px;">Acros Organics</td>
			<td style="width:111px;">424542500</td>
			<td>Toxic, warm at 50 &#176;C to dissolve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tris</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">RDD008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">C4706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">678937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Triton-x100</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">X100</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tween-20</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P9416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tween-80</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P1754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ultra turrax IKA T18 basic tissue homogenizer</td>
			<td style="width:205px;">IKA</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Undec-10-yn-1-ol</td>
			<td style="width:205px;">Fluorochem</td>
			<td style="width:111px;">13739</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Urea</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">U5378</td>
			<td>Toxic, warm at 50 &#176;C to dissolve</td>
		</tr>
	</tbody>
</table>

preparation and in vivo use of an activity-based probe for n-acylethanolamine acid amidase

활동 기반 단백질 프로파일 (ABPP)는 효소의 활성 부위를 타겟팅하는 화학 프로브의 사용을 통해 복잡한 프로테옴 관심 효소의 동정하는 방법이다. 프로브에 도입 된 리포터 태그의 겔 형광 검사, 단백질 오, 형광 현미경, 액체 크로마토 그래피 질량 분석에 의해 효소 표지의 검출을 허용한다. 여기, 우리는 준비 및 복합 ARN14686의 사용, 선택적으로 효소 N -acylethanolamine 산 아미 다제 (NAAA)를 인식하는 클릭 화학의 활동 기반 프로브 (CC-ABP)을 설명합니다. NAAA는 팔미 토일 (PEA)과 oleoylethanolamide (OEA)과 같은 내인성 페 록시 솜 증식 제 - 활성화 수용체 (PPAR) - 알파 작용제를 비활성화하여 염증을 촉진 시스테인 가수 분해 효소이다. NAAA은 리소좀의 산성 pH에서 autoproteolysis에 의해 활성화되는 비활성 전체 길이 proenzyme으로 합성된다. 현지화 연구 시간NAAA은 주로 B 림프구뿐만 아니라, 대 식세포 및 다른 단핵구 유래 된 세포에서 발현되는 것으로 나타 집결지. 우리는 ARN14686 감지하고 단백질 얼룩과 형광 현미경으로 설치류 조직에서 활동 NAAA 생체을 정량화 할 수있는 방법의 예를 제공합니다.

ARN14686는 NAAA 억제제 ARN726의 발판을 기반으로 설계되었다. ARN726의 4-tert- 부틸 시클로 헥 실기가 C9 포화 지방족 쇄 말단 알킨 태그 (도 1) 방위로 치환 하였다. 알킨 태그는 형광 또는 CC 통해 비오틴 분자를 추가하는 두 단계의 표시 절차의 이용을 허용하기 위해 도입되었다. 이 특징은 생체 외 및 생체 내 NAAA을 프로빙 매우 다용도 공구 ARN14686 렌더링한다. 여기, 우리는이 분자의 잠재력을 대표하는 ARN14686의 두 가지 응용 프로그램을 보여줍니다.이 여기에보고 된 실험 절차의 방식이다 그림. 프로브의 정맥 투여 후, 두 개의 서로 다른 검출 방법을 사용할 수 있습니다 단백질 얼룩으로 활성화 NAAA 식의 ⅰ) 분석 및 fluorescenc에 의해 세포 내에서 활성 NAAA 식 및 지역화의 ⅱ) 분석전자 현미경. 첫 번째 대표 결과는 최근 우리 군 (29)에 의해 출판되었다. 우리는 CFA 유도 발 염증의 쥐 모델에서 NAAA 발현을 분석 하였다. 프로브 (3 밀리그램 / kg) 또는 비히클은 나이브 및 CFA 처리 된 래트에서 정맥 주사 하였다. 래트를 4 시간 후에 희생되었다. CC는 프로브 표지 단백질에 비오틴 태그를 소개하고 풍부한 리소좀 추출물에서 수행되었다. 바이오틴 단백질은 다음이었다 스트렙 비즈를 사용하여 농축. 용출 된 단백질은 활성 NAAA의 수준이 현저 대조군의 것과 CFA 대하여 (도 3)로 처리 된 쥐의 발 증가되었음을 나타내는 단백질 블롯에 의해 분석 하였다. 단백질 블롯 분석의 장점은 전기 영동에 의해 분리 된 프로브 반응성 프로테옴의 상세한 검사를 가능하게한다는 것이다. 이 방법은 또한 잠재적 인 프로브 오프 대상의 공개 수 있습니다. 없음의 프로브 컨트롤을 알 수 있어야합니다방법은 실험 배경을 구성 비오틴 내인성 단백질을 배제하기 위해 포함. 도 3의 화살표는 다시 로딩 기준 대조군으로서 사용될 수있다 배경 단백질을 나타낸다. 두 번째 게시되지 않은 실험 (그림 4)에서 우리는 형광 현미경에 의한 생체 검출 NAAA을 조사하기 위해 ARN14686을 사용했다. 우리는 3 ㎎ / ㎏ (IV)에서 마우스에 ARN14686을 투여하고 2 시간 transcardial 재관류에 의한 치료 후에 희생. 폐는 수집라는 접미어, 차가운 2 metylbutane 동결했다. 형광 첨가 대한 CC의 반응을 저온 유지 장치로 수집 된 40 μm의 두께의 조직 슬라이스에서 직접 수행 하였다. 형광 현미경으로 분석 폐포 대 식세포에 속하는 확산 소포 구조의 촉매 활성 NAAA의 존재를 보여 주었다. 단백질 특이 적 항체의 사용 만이 (A)에 비해뽑은 효소 관찰된다. <img alt="그림 1" src="/files/ftp_upload/54652/54652fig1.jpg" /> 그림 1. ARN14686 합성 반응식 운데 10-YN -2- 올 촉매는 4- 디메틸 아미노 피리딘 (DMAP)의 존재 하에서 dipyridylcarbonate (DPC)에 의해 활성화되었다 혼합 카보네이트를 생성한다. 이 탄산은. 표적 분자 ARN14686을 얻을 수있는 아미노산 락탐과 반응시켜 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54652/54652fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54652/54652fig2.jpg" /> 그림 2 :. 본 연구에 나타난 일반적인 전략의 워크 플로우 방식 프로브는 동물 (쥐 또는 마우스)에 주입하고 대상 발현은 두 개의 서로 다른 실험 절차에 따라 분석S : ⅰ) 표지 프로테옴을 추출하고, 비오틴은 CC가 추가됩니다. 스트렙 타비 딘 비드에 바이오틴 단백질의 농축 단계 후, 검색 대상이 단백질 얼룩에 의해 분석된다. ⅱ) 조직의 조각을 준비하고 형광은 CC가 추가됩니다. 검색 대상 현지화는 개화 현미경으로 분석된다. 이 그림은 Bonezzi 등에서 적응하고있다. (29) <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54652/54652fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54652/54652fig3.jpg" /> 그림 3 :. CFA 처리 된 쥐의 발에 NAAA 활성화의 분석 순진한 쥐 (레인 1, 2) 또는 CFA 주입 쥐에서 스트렙 타비 딘 풍부한 단백질 칠일 주입 후의 단백질 얼룩 분석 (레인 3, 4). 쥐 차량이나 ARN14686 (3 ㎎ / ㎏)의 정맥 주사를 받았다. 블로 팅 막형광 스트렙 타비 딘과 프로브했다. 화살표는 NAAA 대역을 나타내고 화살촉은 단백질의 비슷한 금액이 각 레인에로드 된 것을 보여주는, 약 90 kDa의의 비오틴 함유 밴드를 나타냅니다. 이 그림은 Bonezzi 등에서 수정되었습니다. (29) <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54652/54652fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/54652/54652fig4.jpg" /> 그림 4 :. 프로브 표지 형광 현미경으로 마우스 폐에 NAAA 비히클 (A)의 폐 섹션 또는 아 지드 - PEG3 - 플 루어 (545)와 CC 후 ARN14686 주입 (B) 마우스의 대표 사진을보고의 예 생체 감지. V 신호가 검출되지하면서 양 신호 (적혈구) ARN14686 주입 된 마우스에서 검출 된ehicle 투여 마우스. 아 지드 - PEG3 - 플 루어 545 긍정적 인 폐포 대 식세포의 세부 사항은 더 높은 배율로 C에 표시됩니다. 핵은 DAPI (파란색)으로 표시 하였다. 스케일 바 = A에서 50 μm의 C. 10 μm의 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54652/54652fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase at JoVE.com

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

여기서는 활동 기반 프로브의 제조 및 사용을 설명 (ARN14686, 운데 10 ynyl- N - [(3- S) -2- oxoazetidin -3- 일] 카르 바 메이트)의 검출 및 정량화를 허용 염증성 효소 N -acylethanolamine 산 아미 다 아제의 활성 형태 (NAAA), 모두 생체 외 및 생체있다.

준비 및<em&gt; 생체</em&gt; 액티비티 기반 프로브의 사용을위한<em&gt; N</em&gt; -acylethanolamine 산 아미 다제

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

preparation-vivo-use-an-activity-based-probe-for-n-acylethanolamine

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

9.6K 조회수.  Istituto Italiano di Tecnologia.  여기서는 활동 기반 프로브의 제조 및 사용을 설명 (ARN14686, 운데 10 ynyl- N - [(3- S) -2- oxoazetidin -3- 일] 카르 바 메이트)의 검출 및 정량화를 허용 염증성 효소 N -acylethanolamine 산 아미 다 아제의 활성 형태 (NAAA), 모두 생체 외 및 생체있다. 

비디오: Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

양 사체 (Transcriptome) 분석에있는 사용을위한 씨앗에서 매우 정제 된 RNA의 분리를위한 효율적인 방법

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

연구

생화학

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

유 중수 에멀젼의 원심 분리에 의해 거 소포 캡슐화 미소 구의 제조

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

질량 분석 기반 식별 RNA 바인딩 영역에 대 한 샘플 준비

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

각 질의 닭 깃털 및 화장품에의 응용에서의 준비

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

원형 RNA를 분석하기 위해 미니유전자를 포함하는 Alu 요소 사용

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

자동화된 워크스테이션을 사용한 질량 분석법을 위한 플라즈마 시료 준비

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

고품질 발효 생선 제품 준비

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

단백질-지질 상호작용을 측정하기 위한 마이크로스케일 열영동 사용

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

HPLC-MS/MS를 사용하여 뉴클레오시드/뉴클레오티드 함량 측정을 위한 식물 샘플 준비

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...