작업은 저희 연구실은 국립 보건원에서 기금 지원입니다.

준비 <ul><li> Coverslips는 autoclaved해야합니다. </li><li> 멸균 증류수 또는 탈이온수는 솔루션을 준비하고 세탁 coverslips를 위해서 사용해야합니다. </li><li> AC (40 % W / V) 및 BIS - AC (1 % W / V)이 솔루션은 0.2 μm의 여과 소독을하고 있습니다. 빠른 시일 내에 사용하고 살균 필터를하기 전에, 10 % 암모늄 persulfate (100μg/ml 물을 APS)를 준비합니다. APS 솔루션은 매월 바꿉니다. </li><li> autoclaved 수없는 등 3 APTMS, 클로로포름, glutaradehyde, NHS, 그리고 SurfaSil 같은 화학 시약은 hydrogels의 준비를 위해서만 사용되는 할당 병에 보관하고 있습니다. </li><li> 최상의 결과를 얻으려면, hydrogels는 해당 ECM 단백질과 함께 하룻밤 배양 후 며칠 이내에 이용해야합니다. </li><li> 히드로겔의 따르는 이전 50 ML 폴리 프로필렌 팰컨 튜브 클로로포름 (10 ML은 보통 정도로 20 위로 coverslips입니다)의 10 % SurfaSil 솔루션을 준비합니다. (저희 연구실은 보통 0.5 % 글루 타 알데히드 인큐베이션 단계에서 상단 coverslips을 siliconizes). 최소한 10 분 팔콘 튜브와 바위에 coverslips을 추가합니다. SurfaSil 솔루션을 가만히 따르다하고 공기는 hydrogels가 준비되는 생물 학적 안전 캐비닛에 Kimwipes에 coverslips을 건조. </li><li> 다음과 같이 열 - inactivated BSA 솔루션을 준비 : PBS의 지방산 - 초산 무료 BSA의 20 MG / ML 솔루션은 30 분 68 ° C 물 목욕에 incubated입니다. 이 솔루션은 다음 멸균 4 ° C.에 여과 및 저장 </li></ul> 절차 <ol><li> 150 mm 배양 접시의 절반 아랫부분에 Parafilm의 레이어를 놓습니다. </li><li> Parafilm 위에 9 25 mm coverslips까지 플레이스 1 ML 0.1 M NaOH으로 그들을 커버. 진공 라인 대기음 후 3 분 부화합니다. </li><li> 각 coverslip에 화학 후드, 장소 0.5 ML 3 APTMS에서 근무. 3 분위한 부화 후 APTMS를 대기음. 당신이 너무 오래 기다려야하는 경우, 거품이 형성됩니다. </li><li> 같은 접시 20 ML 탈이온수로 번 coverslips을 씻어. 곡선 집게를 사용하여 요리의 coverslips를 제거하고 자신이 대접 측 새로운 150 mm 요리로, 최대 직면와 함께 그들을 전송합니다. 10 분 각 세척 들어, 로커에 탈이온수 세 번있는 coverslips 씻으십시오. 여러분 모두 APTMS을 제거하지 않으면, 그것은 다음 단계에서 글루 타 알데히드와 반응하여 흰색 침전 클라 우디 (그림 1)을 벗어나게됩니다. </li><li> 글루 타 알데히드를 (사용하기 전에 ~ 10 분) 해동. </li><li> 곡선 포셉 사용 Parafilm와 겹쳐 깨끗한 접시에 coverslips를 전송하고 나머지 액체를 대기음. 필요에 따라 나머지 물을 얼룩에 진공 라인이나 Kimwipe을 사용합니다. </li><li> 무균 탈이온수의 0.5 % 글루 타 알데히드 0.5 ML로 완전히 각 coverslip을 커버 및 화학 후드에서 30 분 알을 품다. 글루 타 알데히드를 대기음. 린스와 4 단계에서로 coverslips 씻으십시오. 완전히 coverslips 건조. [당신은 건조한 지역의 중지하고 몇 주 동안 coverslips을 남길 수 있습니다]. </li><li> 당신이 hydrogels를 준비 준비가되면, 생물 안전 캐비닛의 표면에 녹화되어 Parafilm의 시트에 coverslips, 반응 측면까지를 전송 곡선 집게를 사용합니다. coverslips이 Parafilm 표면에 평평하게되었는지 확인합니다. </li><li> 톨루엔의 포화 NHS 솔루션을 준비합니다. (특정 실험에 대한 충분한 톨루엔의 NHS의 작은 금액을 디졸브. NHS가 더 이상 해소하지 않을 때까지 비트에 의해 NHS 비트를 추가하는 유지. 포화 솔루션은 일반적으로 흐리고과 분홍색입니다.) </li><li> 다음, 원하는 아크릴 아미드 비율에 도달하는 아크릴 아미드, 비스 - 아크릴 아미드, 물, APS를 준비합니다. 로 0.8ml의 총 볼륨으로 아래에 설명된 microcentrifuge 튜브의 시약을 추가합니다. </li><li> 그런 다음 한 번에 하나 나누어지는가 NHS를 추가하고 0.8 ML AC 솔루션 TEMED, 소용돌이 간략 즉시 생물 학적 안전 모자 coverslip 당 140 μl를 사용하여 3-5 젤 잔. 가능하다면,이 시점에서 모든 단계는 생물 학적 안전 캐비넷에서 수행되어야합니다. [주 : 서로 다른 크기 coverslips는 필요에 따라 이용하실 수 있습니다. 18 mm coverslips를 사용하는 경우, ~ 33 coverslip 당 μl AC 솔루션과 18 mm 최고 siliconized coverslip을 사용합니다.] <table border="1"><tbody><tr><td></td><td colspan="4"> BIS - AC (%) </td></tr><tr><td></td><td> 0.3 (딱딱한) </td><td> 0.15 </td><td> 0.06 </td><td> 0.03 (소프트) </td></tr><tr><td></td><td> μl </td><td> μl </td><td> μl </td><td> μl </td></tr><tr><td> 물 </td><td> 402 </td><td> 522 </td><td> 594 </td><td> 618 </td></tr><tr><td> AC </td><td> 150 </td><td> 150 </td><td> 150 </td><td> 150 </td></tr><tr><td> 비스 - AC </td><td> 240 </td><td> 120 </td><td> 48 </td><td> 24 </td></tr><tr><td> APS </td><td> 8 </td><td> 8 </td><td> 8 </td><td> 8 </td></tr><tr><td> TEMED</td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> NHS </td><td> 228 </td><td> 228 </td><td> 228 </td><td> 228 </td></tr></tbody></table></li><li> 이 중합을 시작하기 전에 신속하게 각 젤 위에 siliconized 25 mm coverslip을 놓으십시오. 상단 coverslip의 추가는 AC 완전히 바닥 coverslip을 충당하기 위해 허용해야합니다. AC가 polymerizes 때까지 실온에서이 &quot;샌드위치&quot;를 품어. (중합이 발생했을 때 결정하는 microcentrifuge 관의 잔류 AC 솔루션을 확인하십시오. 보통 몇 성실히 젤류 분 동안 부드러운 사람을위한 이상 약간 충분이..) </li><li> 조심스럽게 샌드위치를​​ 픽업 (에 멸균 장갑!)하고 overhangs polymerized 젤 때까지 최고 coverslip을 슬라이드. 그런 다음 젤 그것을 캐내다 수 있습니다. 당신이 최고 coverslip을 제거하기 전에 너무 오래 기다리지는 경우 coverslip가 제거로 젤이 찢어 것입니다. </li><li> 상단 coverslip 폐기하십시오. 물론이 ML PBS /와 함께 6 - 잘 접시에 바닥 젤 - coverslips을 (이하 히드로겔라고도 함) 놓습니다. PBS는 각 잘 수있는 젤 - coverslip 앞 또는 뒤에 추가할 수 있습니다. 로커, 세차 당 5 분에 PBS로 세 번 hydrogels 씻으십시오. </li><li> hydrogels의 필요한 번호가 준비 때까지 반복 11-14 단계를 반복합니다. </li><li> fibronectin 용액 (PBS 3 μg / ML) 또는 기타 ECM 단백질 (클라인 외를 참조하십시오. 자세한 내용은 13.) 2 ML 각 히드로겔 표지. ECM 단백질은 covalently 4 ° C.에서 하룻밤 배양 동안 히드로겔에 바인딩됩니다 </li><li> 37 최소한 30 분 ECM 솔루션과 혈청이없는 미디어 1mg/ml 열 inactivated 지방산 - 초산 무료 BSA와 블록 unreacted NHS를 대기음 ° C 세포 문화 인큐베이터 인치 멸균 PBS 또는 세포 배양 매체로 한 번 hydrogels을 씻어. </li><li> FBS를 포함하는 적절한 문화 매체 플레이트 세포. 히드로겔에 씨앗을 품고 세포의 숫자는 셀 실험에 필요한 확산과 confluency의 정도에 따라 사용자에 의해 결정되어야합니다. 약 10 5 세포는 일반적으로 모래 바닥 서양 qPCR 분석을 위해 충분합니다. </li><li> 잠복기에 따라, 세포의 단백질이나 mRNA가 추출 수 있습니다. coverslips 신중하게 곡선 집게를 사용하여 우물에서 제거하고 실험실 벤치에서 Parafilm의 시트 따라 (2~3cm) 밖으로 간격되었습니다 용해 완충액 100 μl 방울 위에 (세포 쪽 아래) 위치하고 있습니다. (우리는 서쪽 모래 바닥에 대한 샘플을 준비하고 qPCR에 대한 RNA를 분리하고, 각각 표준 SDS 샘플 버퍼와 TRIzol를 사용합니다.) 정확히 하나 분간 용해 완충액으로 세포를 품어. coverslips를 제거하고 microcentrifuge 관에 용해 버퍼를 전송할 수 있습니다. 또는 전용 RNA 추출을 위해, 사용자는 새로운 6 자 판 각 히드로겔를 전송하고 TRIzol / 음의 1ml를 추가할 수 있습니다. 3 분 부화하고 microcentrifuge 관에 저장 TRIzol 솔루션을 제거합니다. </li></ol> 같은 immunofluorescence 같은 절차에 대한 자세한 내용은 hydrogels의 씨앗 세포 BrdU의 얼룩, transfection 등 클라인 외에 설명되어 있습니다. 2007 13. 대표 결과 APTMS의 또한 다음과 같은 coverslips 철저히 세척은 &quot;반응&quot;coverslips 생산에 중요한 단계입니다. 하나는 완전히 APTMS를 제거하지 못하면, 그것은 다음 단계에서 글루 타 알데히드와 반응하여 그림 1A에서 볼 흰색 클라 우디 침전물을 생산합니다. 그림 1B는 제대로 씻어 말린 coverslip을 보여줍니다. 침전물이 개발하는 경우 coverslip가 더 이상 유용한없는 것처럼, 모든 절차는 처음부터 다시 시작해야합니다. 하룻밤 ECM 단백질과 히드로겔 형성과 코팅에 따라, 세포는 다음날 씨앗 수 있습니다. 그림 2 보여줍로서, 뻣뻣한 비해 부드러운 hy​​drogels에 확산 세포 사이에 뚜렷한 차이가있다. 마우스 배아 섬유아 세포 (MEFs)에 phalloidin의 얼룩에서 볼 수 있듯이 소프트 hydrogels에 비해, 전지는 뻣뻣한에 큰 정도로 확산. 사실, 부드러운 히드로겔에 부착 대부분의 전지는 소형 유지하고 덜 효율적으로 연결합니다. 단 MEF의 형태는 그림 2에 표시된 있지만, 세포 확산의 차이가 다른 여러 세포 라인에 걸쳐 일관가 11-12,14을 테스트했습니다. 성공의 비밀 <ol><li> 절차가 시작되면, 그것은 &quot;반응&quot;면을 coverslips를 유지하고 코팅되어있는 면을 염두에 두어야하는 것은 매우 중요합니다. </li><li> 장갑은 가능한 한 무균되는 작업 환경을 제공하기 위해 절차를 수행하는 동안 모든 시간에 착용한다. </li><li> 초기 단계의 대부분은 화학 퓸 후드 및 실험실 벤치 사이에 수행됩니다. coverslips에서 여분의 글루 타 알데히드를 제거하기 위해 이후 모든 단계는 생물 학적 안전 캐비닛에 따라 실행해야합니다. </li><li> 세포 sprea의 차이로 인해뻣뻣한 hydrogels에 비해 땡 (그림 2), 우리는 부드러운 hy​​drogels에 대한 세포의 두 수를을 심는 것이 좋습니다. </li><li> 교류 중합 과정은 매우 빠르다. 처음 사용자의 경우, 하나는 APS의 양을 줄일 수 또는 중합 과정을 연장하는 솔루션 TEMED. 한 번에 몇 coverslips 이상을 준비하려고하지 마십시오. </li><li> G0에서 동기화가 필요한 세포주기 연구에서 우리는 일반적으로 플라스틱 문화 요리 세포를 혈청 - 굶어 세포를 trypsinize하고 mitogens의 존재 (FBS 및 / 또는 성장 요소) 다른 compliances의 hydrogels 그들을 reseed. 이 절차는 G0 - 동기 세포의 시작 인구가 모든 샘플에서 동일 보장합니다. 그러나 많은 신호 전달 연구를 위해, 필요한 mitogen 자극의 기간은 첨부 파일 및 세포의 확산을위한 충분한 길이되지 않을 수 있습니다. 이 경우에, 우리는 hydrogels에있는 세포를 혈청 - 굶어 후 직접 mitogen으로 그들을 자극. </li></ol><img src="/files/ftp_upload/2089/2089fig1a.jpg" alt="그림 1A" /> 그림 1A. 부적절 coverslip을 빨기도 했죠. APTMS의 또한 다음, coverslip는 글루 타 알데히드 솔루션뿐만 아니라 전 1~2분에 대한 졌죠. 침전물이 절차에 설명된 단계에 따라 씻어되지 않은 coverslip을 형성한다. <img src="/files/ftp_upload/2089/2089fig1b.jpg" alt="그림 1B" /> 그림 1B. 적절히 coverslip 세탁. APTMS의 다음 또한이 coverslip는 글루 타 알데히드 솔루션뿐만 아니라 전 세 번 십분마다 세탁했습니다. 아무도 침전은 coverslip에 형성하지 않습니다. <img src="/files/ftp_upload/2089/2089fig2.jpg" alt="그림 2" /> 그림 2. 강성 변화.의 hydrogels에 대한 세포의 형태 MEFs가 9시간에 대한 높거나 낮은 강성의 fibronectin - 코팅 hydrogels에 씨앗을 품고 있었다 설립. 잠복기에 따라, 세포는 고정 per​​meabilized 및 F - 굴지에 바인딩 FITC - phalloidin 물들일되었습니다. 높은 강성 젤에 MEFs 낮은 강성 hydrogels에 씨뿌린 사람에 비해 스트레스 섬유를 전시하고 잘 확산됩니다. 스케일 바 = 50μm. <img src="/files/ftp_upload/2089/2089fig3.jpg" alt="그림 3" /> 그림 3. 마우스 cyclin의 D1의 mRNA 수준의 대표 양적 PCR 결과. 세럼 굶주린 생쥐 배아 섬유아 세포가 높은 (3 % 아크릴 아미드) 또는 낮은 (0.3 % 아크릴 아미드) 강성 hydrogels에 도금 9 시간 10 % FBS와 자극했다. RNA 추출 다음, 실시간 정량 PCR 분석은 cyclin D1의 mRNA의 수준 (18S RNA에 정규화)을 수행했습니다. G0가 정지 세포에서 cyclin D1의의 mRNA를 나타냅니다. Cyclin D1의 mRNA의 수준이 뻣뻣한 hydrogels에 아니지만 부드러운 hy​​drogels을 크게 향상시킬 수 있습니다. SD 중복 PCR 반응의 - 데이터 + /을 의미합니다.

<ol>
	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Duprez, D. A., Cohn, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+mechanical+properties+of+human+atherosclerotic+tissue+by+high-frequency+intravascular+ultrasound+imaging.+An+in+vitro+study.">Prediction of mechanical properties of human atherosclerotic tissue by high-frequency intravascular ultrasound imaging. An in vitro study.</a> Arterioscler Thromb. 12, 1-5 (1992).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples.">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples.</a> Phys Med Biol. 52, 1565-1576 (2007).</li><li>Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+stiffness+in+regulating+cell+behavior.">The role of matrix stiffness in regulating cell behavior.</a> Hepatology. 47, 1394-1400 (2008).</li><li>Pelham, R. J., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc. Natl. Acad Sci USA. 94, 13661-13665 (1997).</li><li>Klein, E. A., Yung, Y., Castagnino, P., Kothapalli, D., Assoian, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion,+cellular+tension,+and+cell+cycle+control.">Cell adhesion, cellular tension, and cell cycle control.</a> Methods Enzymol. 426, 155-175 (2007).</li><li>Klein, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle+control+by+physiological+matrix+elasticity+and+in+vivo+tissue+stiffening.">Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening.</a> Current Biology. 19, 1511-1518 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

히드로겔 중합 과정의 중요한 요소는 세포가 아니라 ECM - 코팅 히드로겔 자체보다 유리 coverslip에 바인딩 수 있도록 공기 방울 형성​​을 방지하는 것입니다. 이것은 vortexing 시각도 기포가 젤에 갇혀가 없습니다 중일 후 신중하게 중합 솔루션을 pipetting하여 예방할 수 있습니다. 우리는 항상 실험에 대한 충분한 발생시키기 위해 추가 &quot;반응&quot;coverslips 및 hydrogels을 준비하는 것이 좋습니다. </p...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
</tr>
<tr>
<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
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studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

조직 강성은 세포 기능의 중요한 결정자이며, 조직 강성의 변화는 일반적으로 섬유증, 암 및 심장 혈관 질환 1-11와 관련된 있습니다. 세포 기능을 공부하기 위해 전통적인 세포 생물 학적 접근은 탄성 ECM 또는 조직 사이의 ECM의 강성의 변화의 영향에 대해 설명할 수없는 단단한 하층에 culturing 세포를 (플라스틱 요리 또는 유리 coverslips) 포함. 체외에서 생체내 조직 준수 조건 모델에, 우리와 다른 ECM - 코팅 hydrogels을 사용합니다. 우리 연구실에서 hydrogels는 생물학 12 본 조직 compliances의 범위를 모방 수 polyacrylamide를 기반으로합니다. &quot;반응&quot;커버 전표는 3 APTMS의 또한 다음 NaOH와 인큐베이션에 의해 생성됩니다. 글루 타 알데히드는 3 APTMS와 polyacrylamide 젤을 상호 연결하는 데 사용됩니다. 아크릴 아미드 (AC), 비스 - 아크릴 아미드 (BIS - AC)과 암모늄 persulfate의 솔루션은 히드로겔의 중합에 사용됩니다. N - hydroxysuccinimide (NHS)이 히드로겔에 crosslink ECM 단백질에 AC 솔루션에 통합됩니다. 히드로겔의 중합에 따라, 겔 표면은 같은 fibronectin, vitronectin, 콜라겐 등 선택의 ECM 단백질로 코팅되어 히드로겔의 강성은 레올로지 또는 원자 힘 현미경 (AFM)에 의해 결정 및 솔루션 12 AC 및 / 또는 BIS - AC의 비율을 변화하여 조정할 수 있습니다. 이러한 방식으로, 하층의 강성도 레올로지 또는 AFM을 사용하여 계량 수있는 생물 학적 조직의 강성 일치될 수 있습니다. 세포 그런 다음 필요한 실험 조건에 따라 이러한 hydrogels과 교양에 씨앗을하실 수 있습니다. 분자 분석을위한 세포 및 복구의 영상은 간단합니다. 이 기사에 대한, 우리는 20,000 파스칼 &lt;E 분들로 3000 파스칼과 치열한 substrata / 조직&gt; 탄성 moduli (E)를 가지고 그 같은 부드러운 substrata을 정의합니다.

Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

세포 기능에 substrata의 강성의 효과는 모델 수<em&gt; 체외에서</em&gt; compliances 변화의 polyacrylamide의 hydrogels을 사용합니다.

아크릴 아미드 기반 Hydrogels을 사용하여 세포 기능에 매트릭스 강성의 영향을 공부

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

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21.9K 조회수.  University of Pennsylvania. 세포 기능에 substrata의 강성의 효과는 모델 수 체외에서 compliances 변화의 polyacrylamide의 hydrogels을 사용합니다.

비디오: Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

조직 공학을위한 3D Hydrogels에서 셀룰러 봉지

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

연구

생물학

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

광학 트랩을 사용하여 박테리아 세포의 굽힘 강성을 측정

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

내생 와일드 타입 BRCA1을 표현 세포를 사용하는 동종 재조합에 BRCA1 변이의 효과를 확인

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

편광 상피 세포에 Plasmids의 Microinjection에 의해 작은 GTPases의 기능을 분석

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

시토크롬를 사용 Mitochondrial 호흡 기능의 시각화 C 데이스 / 호박산 탈수소 효소 (COX / SDH)를 두 번 라벨 Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

에서 Hypoxia의 효과를 연구하기 위해 정의된 기체 환경 만들기<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

에 박테리아의 효과를 측정<em&gt; C. 엘레</em계란 보존 분석을 사용하여&gt; 행동

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Zebrafish 독도에서 단일 셀 해상도 칼슘 이미징 사용 하 여 베타 세포 기능 분석

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

아디포세포 기능에 대한 단백질 및 마이크로 RNA 변조의 영향을 연구하는 세포 배양 모델

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

시험관 내 E3 유비퀴틴 리가제 기능 분석

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...