우리는 사용 된 마우스 척수 조직 샘플을 제공하기 위해 미네소타 대학의 리 실험실에게 감사의 말씀을 전합니다. 이 작품은 MB의 랜디 면도기 암 연구 및 지역 사회 기금과 미네소타 난소 암 연맹 (MOCA)이 MB의에 의해, 국방 난소 암 연구 프로그램 (OCRP) MB의 OC093424의 부서에 의해 지원되었다. 자금 제공자는 연구 설계, 자료 수집 및 분석, 게시하는 결정 또는 원고의 준비를 전혀 역할을 없었다.

1. 용해 버퍼 준비 <ol><li> 2 M 원액을 만들기 위해 탈 (DI) 물에 설탕을 녹인다. 0.22 ㎛의 폴리 불화 비닐 리덴 (PVDF) 필터를 구동 진공을 사용하여 수크로오스 2 M 용액 필터. </li><li> 혐기성 조건에서 500 mM의 스톡 용액을 저장하기 위해 DI 물에 디티 오 트레이 톨 (DTT)을 녹인다. 100 mM의 스톡 용액을 제조하기 위해 DI 물에 염화 마그네슘 (의 MgCl 2)을 녹인다. 50 mM의 스톡 용액을 제조하기 위해 DI 물에 아데노신 삼인산 (ATP) 나트륨 수화물을 녹인다. </li><li> DI 물에 후, Trizma 염산염을 용해시키고 수산화 나트륨 (NaOH)을 이용하여 pH를 조정하여 pH를 7.4 트리스 용액을 구성한다. </li><li> 250 mm의 1mm를 5mm의의 MgCl 2 농도 1mM이 ATP 농도 및 50 mm의 트리스 농도 DTT 농도의 슈 크로스 농도의 용해 완충액을 만들기 위해 DI 물에 원액의 일부를 겸용 . 예를 들어, 트리스, 50 μL를 혼합 535.30의 MgCl 2 μL, 1.25 수크로오스 ㎖, 20 μL의 DTT, ATP 200 μL 및 DI 물 7.4947 ml의 용해 완충액 10 mL로한다. 이것은 약 20 실험에 사용될 수있다. </li></ol> 실험 2. 배양 세포 <ol><li> DMEM + 10 %, 50㎖ 태아 소 혈청 (FBS) 원추형 튜브의 30 ㎖를 분취하여 미디어를 확인. </li><li> 미지근한 물을 작은 비커에 액체 질소 저장에서 냉동 M17이나 헬라 세포를 전송합니다. </li><li> 50 ML 원뿔 튜브에 세포를 전송합니다. 첫째, 세포 바이알에 일부 미디어를 추가합니다. 그리고 해동 5 초 이내에 재현 탁 세포를 전송합니다. </li><li> 세포 펠렛을 얻기 위해 5 분 동안 450 XG에 세포 현탁액을 원심 분리기. T75 플라스크에 매체의 20 ML을 추가합니다. </li><li> 흡입을 사용하여 세포에서 용지를 제거합니다. 세포 펠렛 및 재현 탁에 신선한 매체의 5 ML을 추가합니다. </li><li> T75 플라스크에 미디어의 20 ㎖에 5 ml의 세포 현탁액을 전송하고 37 ° C에서 품어. 채널3 일마다 미디어를 앙. </li></ol> 3. 셀 수확 <ol><li> 세포를 만지지 않도록 조심하면서, 흡입을 통해 용지를 제거합니다. </li><li> 인산염 완충 생리 식염수 5 ㎖ (PBS)와 세포를 씻으십시오. 흡입을 사용하여 PBS를 제거합니다. </li><li> T75 플라스크에 트립신 에틸렌 디아민 테트라 아세트산 (EDTA) 3 ㎖를 추가하고 3 분 동안 37 ℃에서 배양한다. </li><li> 플라스크에 신선한 미디어 6 ML을 추가하고 세포를 재현 탁. </li><li> 5 분 동안 290 × g에서 15 ML 원뿔 튜브와 원심 분리기에 현탁액을 전송합니다. </li><li> 상층 액을 제거하고 PBS 5 mL를 추가 할 수 있습니다. 조심스럽게 펠렛을 분쇄하기 위해 튜브의 바닥을 누릅니다. 5 분 동안 290 XG에 원심 분리기. </li><li> 단계를 반복 3.6 세 번. </li><li> PBS를 제거하고 신선한 PBS 1 ㎖로 세포를 재현 탁. 에펜 도르프 튜브 (1.5 ㎖)에 전송합니다. 5 분 동안 290 XG 스핀. </li></ol> 4. 세포 용해 <ol><li> 피펫을 사용하여 펠릿의 대략적인 체적을 측정1 부피비 : 질량에 유리 비드를 달아 (D1). 용해 완충액의 펠릿 두번 볼륨. 참고 : 유리 구슬을 추가하기 전에 버퍼를 추가합니다. </li><li> 를 Lyse 냉장실의 텍싱하여 4 ° C에서 30 분 동안 교반 최대의 에펜 도르프 튜브에 세포. </li><li> 200 XG 30 초 원심 분리기 짧게 구슬을 해결합니다. 상층 액을 수집합니다. </li><li> 상층 액에 이전과 유리 구슬의 동일한 볼륨을 추가합니다. </li><li> 다시 한번 최대 교반에서 4 ℃에서 30 분 동안 소용돌이. </li><li> 200 XG 30 초 원심 분리기 짧게는 유리 구슬을 해결합니다. 상층 액을 수집합니다. </li><li> 5 분 5,030 XG에 원심 분리기는 핵, 세포막 손상되지 않은 세포를 제거합니다. 세포 용 해물 인 상층 액을 수집합니다. </li></ol> 5. 조직의 균질화 <ol><li> 용해 버퍼에 마우스 척수 조직의 9 해결책 : 1의 부피 : 질량을 확인합니다. 참고 :이 방법은 다른 다양한에 적용 할 수있다ENT 조직 샘플. </li><li> 남아있는 청크가 없다는 것을 보장하기 위해 30 초 동안 균질 기에서 2 레벨의 설정을 사용하여 조직을 균질화. </li><li> 핵, 세포막 손상되지 않은 세포를 제거하기 위해 3 분 동안 5,030 XG에 스핀 다운. </li><li> 해물 인 상층 액을 수집합니다. </li></ol> 6. 샘플 유도체 화 <ol><li> 피어스 BCA 단백질 분석 키트 프로토콜에 의해 지정된대로 비색 96- 웰 플레이트 판독기를 사용하여 조직 파쇄 액의 단백질 농도를 결정하기 위해 96 웰 플레이트 분석 bicinchoninic 산 (BCA)을 수행한다. </li><li> 탈 이온화 (DI) 물을 사용하여 50 μL에 총 단백질의 20 μg의에 해당하는 나누어지는 가져와. </li><li> 솔루션 HA-UB-비닐 설폰 (VS)의 1.35 μm의 2 μl를 추가하고 37 ℃에서 1 시간 동안 배양한다. 이것은 최종 반응 혼합물에 프로브를 50 nm의 용 해물 20 μg의 결과. 이 더빙 기능들의 태깅을 위해 큰 몰 과량이다. </li><li> 나는95 ° C에서 5 분간의 램 믈리 시료 완충액에서 샘플 ncubate. 52 ㎕의 반응 부피로 사용 배 샘플 완충액 26 μL, 4X 샘플 완충액 13 μL 또는 6X 샘플 완충액 8.87 μL를 들어. </li><li> 젤을로드하기 전에 얼음 쿨. </li></ol> 7. 서쪽 오점 <ol><li> 준비된 샘플의 40 μL와 12 웰 4-20% 트리스 - 글리신 젤로드 </li><li> 이온 전면이 바닥에 도달 할 때까지 95 V에서 젤을 실행합니다. </li><li> 폴리 비닐 리덴 디 플루오 라이드 (PVDF) 멤브레인 상에 겔의 하룻밤 전송을 수행합니다. </li><li> 아미 검은 얼룩의 막을 인큐베이션하고 각 레인에있는 단백질의 양을 보여주는 화상을 얻을 membane 스캔. </li><li> 막 Destain 및 차단하는 1 시간 동안 PBS에 5 % 우유에 품어. </li><li> 1의 농도 차 항 HA 항체에 막을 인큐베이션 : 어느 밤새 4 ℃에서 또는 실온에서 4 시간 동안 PBS에서 10,000 5 % 우유. </li><li> f를 3 세척을 수행0.1 % 트윈 20 세제와 PBS에 5 분마다. </li><li> 1의 농도로 마우스 고추 냉이 퍼 옥시 다제에 막을 인큐베이션 : PBS 10,000 5 % 우유를 적어도 2 시간 동안. </li><li> 0.1 % 트윈 20 세제와 PBS에 5 분마다 3 세척을 수행합니다. </li><li> PBS에서 5 분 동안 1 세척을 수행합니다. </li><li> 10 분 동안 화학 발광 검출 시약에서 부화 막과 화학 발광 검출기를 이용하여 검출한다. 검출기에 자동 노출 설정을 사용합니다. </li></ol>

<ol>
	<li>Ovaa, H., Kessler, B. M., Rol&#233;n, U., Galardy, P. J., Ploegh, H. L., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-+based+ubiquitin-specific+(USP)+profiling+of+virus-infected+and+malignant+human+cells.">Activity- based ubiquitin-specific (USP) profiling of virus-infected and malignant human cells.</a> Proc Natl Acad Sci USA. 101 (8), 2253-2258 (2004).</li><li>Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination:+Targeting+of+proteins+for+degradation+by+the+proteasome.">Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome.</a> Semin Cell Dev Biol. 11 (3), 141-148 (2000).</li><li>Ziad, M. E., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+proteolysis+by+human+deubiquitinating+enzymes.">Regulation of proteolysis by human deubiquitinating enzymes.</a> Biochim Biophys Acta. 1843 (1), 114-128 (2014).</li><li>Rol&#233;n, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+Profiling+of+Deubiquitinating+Enzymes+in+Cervical+Carcinoma+Biopsies+and+Cell+Lines.">Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines.</a> Mol Carcinog. 45 (4), 260-269 (2006).</li><li>Borodovsky, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry-Based+Functional+Proteomics+Reveals+Novel+Members+of+the+Deubiquitinating+Enzyme+Family.">Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family.</a> Chem Biol. 9 (10), 1149-1159 (2002).</li><li>Andersson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Parkinson&#8217;s-disease-associated+mutations+on+the+deubiquitinating+enzyme+UCH-L1.">The effect of Parkinson&#8217;s-disease-associated mutations on the deubiquitinating enzyme UCH-L1.</a> J Mol Biol. 407 (2), 261-272 (2011).</li><li>Poon, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Amyloid+(A&#946;)+oligomers+impair+brain-derived+neurotrophic+factor+retrograde+trafficking+by+down-regulating+ubiquitin+C-terminal+hydrolase+UCH-L1.">&#946;-Amyloid (A&#946;) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase UCH-L1.</a> J Biol Chem. 288 (23), 16937-16948 (2013).</li><li>Nijman, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP1+regulates+the+Fanconi+anemia+pathway.">The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.</a> Mol Cell. 17 (3), 331-339 (2005).</li><li>Stevenson, L. F., Sparks, A., Allende-Vega, N., Xirodimas, D. P., Lane, D. P., Saville, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP2a+regulates+the+p53+pathway+by+targeting+Mdm2.">The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.</a> EMBO J. 26 (4), 976-986 (2007).</li><li>Coughlin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+RA-9+inhibits+proteasome-associated+DUBs+and+ovarian+cancer+in+vitro+and+in+vivo+via+exacerbating+unfolded+protein+responses.">Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.</a> Clin Cancer Res. 20 (12), 3174-3186 (2014).</li><li>Colla, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+&#945;-Synucleinopathy+In.">Endoplasmic Reticulum Stress Is Important for the Manifestations of &#945;-Synucleinopathy In.</a> Vivo. J Neurosci. 32 (10), 3306-3320 (2012).</li></ol>

저자가 공개하는 게 없다.

유비퀴틴은 기본적인 셀룰러 활성이기 때문에, 규제 메커니즘을 이해하는 것이 질병의 병리 과정을 발굴하는 키가 될 수있다. HA의 사용은 여기에보고 UB 유래 활성 부위 감독 프로브는 UB-매개 단백질 분해 연구를위한 쉬운,하지만 매우 적용 할 수있는 방법을 제공하는 태그. 이 방법은 신속하고 저렴 개별적 더빙 각각 공부보다 길다. 유리 비드를 사용하는 -이 방법에서, 세?...

유비퀴틴 - 프로 테아 좀 시스템 (UPS)은 포유 동물 세포에서 주요 분해 경로의 하나로서 역할을한다. 프로 테아 좀 저하 행 기판을 공유 유비퀴틴 (UB) 1 중합체와 태그. 타겟 기판 분해 테아로 들어가기 전에, 폴리 - 유비퀴틴 태그는 제거되어야한다. 효소 (더빙) deubiquitinating 알려진 효소 클래스 유비퀴틴 분자 (2)의 제거 및 재활용 할 책임이있다. 이는 셀 3의 백 작업 거의 더빙이 있다는 인간 게놈으로부터 예측되어왔다. UB-매개 세포 과정을 조절 움직여 같은 많은 수의,이 효소를 공부에만 발현 수준에 대한 정보를 제공 mRNA의 기술 활동 및 서부 블로 팅에 대한 정보를 제공하지 않기 때문에 문제를 제시한다. 인플루엔자 헤 마글 루티 닌 (HA)의 사용은 공유 개조를 허용 활성 부위 지향 프로브 UB 유래, 태그기능 더빙 따라서의 형 치수는 웨스턴 블롯 4에이 효소의 활동의 직접적인 시각화를 제공합니다. 프로브는 활성 부위 시스테인 잔기 5 자살 기질로서 작용 C 말단 티올 반응성기를 갖는다. 이러한 프로브, 상기 셀의 양쪽 병리 생리 학적 상태에 따라 다수의 더빙의 활성 및 발현 가능성을 연구 할 수있다. DUB 활성의 변화는 파킨슨, 알츠하이머, 빈혈 및 각종 암 6-10 같은 병리 적 상태의 범위에 연루되어왔다. 이 기술은 질병의 연구를위한 강력한 도구를 제공합니다. 본 논문에서는 유리 비드를 사용하여 용해 된 헬라 세포 및 M17에서이 기술의 애플리케이션을 도시한다. 또한, 우리는 마우스 척수 조직 샘플에서이 방법을 사용하는 방법을 설명합니다. 이 방법에서 얻은 정보를 식별하기위한 출발점으로 사용될 수있다치료 대상뿐만 아니라 다른 질병 상태의 연구를위한 모델을 확립. 이 기술의 진정한 유틸리티는 단일 분석에서 여러 더빙에 대한 정보를 제공 할 수있는 능력에있다.

<table border="0" cellpadding="0" cellspacing="0" style="width:615px;" width="615">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PowerGen 125 ( Homogenizer)</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">14-261-02P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum-driven filters .22&#181;M&#160;</td>
			<td style="width:113px;"></td>
			<td style="width:119px;">BPV2210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed &#8804;106&#181;m (~140 U.S. sieve)</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">G4649-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">S6-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adenosine 5&#39;-triphosphate disodium salt hydrate</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">A26209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma hydrochloride&#160;</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">T3253</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture flask 75cm2 w/ filter cup 250 ml 120/ cs</td>
			<td style="width:113px;">Cellstar</td>
			<td style="width:119px;">T-3001-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HI FBS</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">16140-071</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-HA antibody produced in mouse</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">H9658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ubiquitin vinyl sulfone (HA-tag)</td>
			<td style="width:113px;">Enzo Life Sciences</td>
			<td style="width:119px;">BML-UW0155-0025</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laemmli&#39;s SDS-Sample Buffer (4X, reducing)</td>
			<td style="width:113px;">Boston BioProducts</td>
			<td style="width:119px;">BP-110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:113px;">ThermoScientific</td>
			<td align="right" style="width:119px;">23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

유비퀴틴 - 프로 테아 좀 시스템은 최근 신경 변성 질환 및 암을 비롯한 다양한 병리에 관여한다. 이러한 관점에서,이 시스템의 조절 메커니즘을 연구하는 기술은 전술 한 질환의 세포 및 분자 적 프로세스를 해명에 필수적이다. 이 논문에 설명 된 적혈구 응집소 유래 유비퀴틴 프로브의 사용은이 시스템의 연구를위한 유용한 도구로서 기능한다. 이 논문은 효소 활성을 deubiquitinating의 직접적인 시각화를 제공 분석을 수행 할 수 있도록 해주는 방법을 자세히 설명합니다. Deubiquitinating 효소는 프로 테오 열화를 제어하고, 사용자가 하나의 분석에서 여러 효소의 활성을 조사 할 수 있도록 자신의 활성 부위에서 기능적 유사성을 공유한다. 해물을 부드러운 기계 세포 파괴를 통해 얻은 액티브 사이트 감독 프로브와 함께 배양한다. 비활성 효소가 결합되지 않은 유지하면서 기능 효소는 프로브와 태그. 실행으로닝이 분석에서, 사용자는 활성 및 빠르고 손쉽게 방식으로 여러 deubiquitinating 전위 효소 발현 모두에 대한 정보를 획득한다. 현재의 방법은 인간 세포에서 예측 백 deubiquitinating 효소 개별 항체를 사용하는 것보다 훨씬 더 효율적이다.

배양 된 M17과 헬라 세포는 프로토콜 (3 셀 수확)에 자세히 방법 및 마우스 척수 조직을 얻었다을 사용하여 수확했다. 세포 펠렛 / 척수 조직은 시약 제조 섹션에 기재된 용해 완충액으로 튜브에 넣었다. 세포 펠릿을 균질화 (도 1b)를 이용하여 균질화시켰다 유리 비드 (도 1a) 및 마우스 척수 조직 샘플을 사용하여 용해시켰다. 용해 또는 균질화 한 후, 샘플을 유리 비드 및 / ?...

Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

현재 프로토콜은 기능적 상동 deubiquitinating 효소의 활성을 측정하는 방법을 자세히 설명. 전문화 된 프로브에 공유 효소를 검출하고 수정할 수 있도록. 이 방법은 새로운 치료 표적을 식별하는 잠재력을 보유하고있다.

세포주 및 조직 샘플에서 Deubiquitinating 효소의 활성을 측정 방법

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

method-for-measuring-activity-deubiquitinating-enzymes-cell-lines

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JoVE Journal

Biology

9.5K 조회수.  University of Minnesota.  현재 프로토콜은 기능적 상동 deubiquitinating 효소의 활성을 측정하는 방법을 자세히 설명. 전문화 된 프로브에 공유 효소를 검출하고 수정할 수 있도록. 이 방법은 새로운 치료 표적을 식별하는 잠재력을 보유하고있다. 

비디오: Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

연구

생물학

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

조직 샘플에서 γH2AX Foci의 Quantitation

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

의 활동을 측정하는 어세이 대장균은 라이신 Decarboxyase을 Inducible

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

자질<em&gt; 체외</em효소 특성에 대한&gt; ATP 아제 활동

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

유방암 세포주에서 세포 증식을 결정하기위한 세 가지 방법 비교

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

하이브리드 컷 : 난 분해성 식물 조직 샘플에 대한 개선 단면 방법

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

출생 후의 쥐의 청각 수포의 해부 : 중이의 뼈와 조직 학적 분석의 분리

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-지우기: 조직 지방 조직의 3 차원 이미징 방법 삭제

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...