우리는 기술 지원 Zehavit Frenkel와 산드라 Tolchinsky 감사드립니다. 이 작품은 관련 연구는 이스라엘 과학 재단 (1229년에서 1207년까지)과 독일 - 이스라엘 프로젝트 협력 (DIP - DFG)에서 기금에 의해 지원됩니다.

다음 프로토콜은 총 glycoproteins이나 관심의 특정 당단백질의 설탕 체인 분석위한 것입니다. 프로 시저는 기본적으로 프로토콜을 통해 언급한 몇 가지 변경 사항과 함께 두 경우 모두에 대해 동일합니다. <ol><li> N - 연결 glycans의 대사 라벨링을 위해 각 샘플에 대해 90mm 접시에 밤새 자란 포유 동물 세포의 subconfluent 문화를 사용하는 한 요구 (샘플 서로 다른 시간 지점이나 트리 트먼트를 나타내는 수 있습니다.) 이 프로토콜은 NIH 3T3 세포에 최적화되어 있습니다. </li><li> 10% dialyzed FCS와 5-15분 4 MM 나트륨 pyruvate와 함께 제공된 신선한 사전 예열 (37 ° C) 포도당없는 매체, 5 ML에서 부화하여 포도당에 대한 세포를 굶어. </li><li> 사전 예열 1 ML과 기아 매체를 교체 (37 ° C) 포도당 무료 400 μCi가 포함된 매체 [2 - 3 H] (낮은 65 농도 μCi / ML은 총 glycoproteins의 라벨에 사용됩니다) mannose - 라벨, 1 시간 동안 정상적인 조직 문화 조건에서 세포를 품어. </li><li> 각 샘플의 라벨 미디어를 제거하고, 신중하게 4 PBS 2 ML을 추가 ° 추적에 해당하는 샘플을 필요로하는 동안 맥박과 얼음 (이 샘플 펄스라고도합니다) 그들을 장소에 해당하는 샘플을 C, 2 ML 3 번 씻어서 미리 예열 (37 ° C) 정상 완료 배지, 그리고 37 CO 2 배양기에 배치 ° C 원하는 추적 기간 동안 미리 예열 정규 매체의 5 ML과 함께. </li><li> 얼음처럼 차가운 PBS 2 ML과 펄스 샘플 (이전 얼음에 위치) 3 번 린스. 세포는 다음 셀 스크레이퍼를 사용하여 두 ML PBS에서 요리를 긁어 냈어요, 그리고 2 ML Eppendorf 튜브에 배치됩니다. </li><li> 16000Xg (60-10 초)에있는 세포 단기 스핀, 표면에 뜨는를 버리고 -80에서 세포 펠릿 ° C.를 동결 </li><li> 단계 5 체이스 시간의 끝에 추적 샘플에도 6 수행합니다. </li><li> 냉동실에서 세포를 제거하고 버퍼 300 μl를 추가하여 그들을 lyse은 잠시을 vortexing하고 부화하여 버퍼 B와 함께 얼음에 20 분 얼음 20 분 잠복기, 또는 총 glycoproteins 분석의 경우, sonication 다음 (4 회, 10 초, 최대 진폭), 다음 5 분에 대한 샘플을 끓여. </li><li> 4 ° C. 20 분 16000Xg에서 lysates을 원심 분리기 새로운 Eppendorf 튜브에 뜨는을 전송하여 펠렛을 삭제. </li><li> 총 glycoproteins를 분석하면, 분자 여과 및 deglycosylation (단계 17)로 진행합니다. H2a의 당단백질의 immunoprecipitation 여기 시연에 대한 (우리의 예를 들어 안티 H2a 항체에 (Repligen) 단백질 A - 아가로 오스 비즈 1시 1분 정지하고 원하는 당단백질를위한 토끼 polyclonal 방지 H2a 항체 3 μl / 샘플 20l/sample을 추가 ), 9 단계에서 뜨는 있습니다. </li><li> 지속적으로 속도가 느린 회전에 의해 혼합, 4 ° C에서 4-16시간에 대한 샘플을 품어. </li><li> 30 초 동안 16000Xg에서 비즈를 스핀 다운, 그리고 신중하게 뜨는 사용하여 진공을 제거합니다. </li><li> 500 μl 버퍼 D를 추가하고 vortexing하여 구슬을 씻어. 12 단계에서와 같이 회전하여 표면에 뜨는를 제거합니다. 세차 3 회 반복합니다. </li><li> 비드 펠렛에 10μl denaturing 버퍼 (코 엔도 - H 키트와 함께 제공되는) 추가하고 5 분 (총 단백질을 분석하는 경우, deglycosylation가 Microcon 필터 단계 17을 수행합니다)에 대한 샘플을 끓여. </li><li> (30 초 동안 16000Xg) 비즈를 스핀 다운, 새로운 Eppendorf 튜브에 뜨는를 전송합니다. 다음 엔도 H 효소의 0.5μl는 반응 버퍼 (도 코 엔도 H 키트와 함께 제공) 10 μl와 함께 각각의 샘플에 추가되며, 샘플은 3 시간 동안 ° C 37 incubated 수 있습니다. </li><li> 단백질에서 릴리스된 glycans을 분리하려면 샘플은 다음 3 분 14000Xg에 centrifuged, 컷 - 오프 30kDa와 탈이온수 5 번 희석 및 분자 필터 (Microcon Ultracel YM30)의 상단에 위치합니다. 탈이온수 및 샘플 반복 원심 분리의 50μl을 적용합니다. 이 워시 아웃은 흐름을 통해 유지, 2 번 더 반복합니다. </li><li> 당신이 총 glycoproteins을 분석하는 경우 Microcon 필터에 lysates의 뜨는을 (9 단계에서) 적용됩니다. 3 분위한 14000Xg에서 반복 원심 분리하여 3 번 버퍼 E, 100 μl로 추출 씻으십시오. 10X 엔도 H 반응 버퍼 3 μl와 Microcon retentate에 엔도 H 효소 1.5 μl를 추가하고, 37 3 시간 동안 품어 ° C. 공개 glycans는 단계 16로 3 반복 centrifugations의 탈이온수와 eluted 있습니다. </li><li> 원하는 경우, 엔도 H 방지 glycoproteins를 포함하는 단계를 16 17 retentates은 다음 37, 버퍼 C 15 μl에 N - glycosidase F 조의 200mU와 incubated 아르 ° C를 16 H.위한 탈이온수과 용출은 단계 16로 이루어집니다. </li><li> Speed​​Vac 농축기에서 엔도 H 반응 흐름을 통해이 (단계 16 17)가 들어있는 튜브를 배치그리고 완전히 샘플을 건조 (이것은 45까지 할 수있는 ° C는이 과정을 가속). </li><li> HPLC에 대한 샘플을 준비하려면, HPLC 용매 (물, 60:40, 1 %의 인산 acetonitrile) 12 μl의 건조 알약을 resuspend. </li><li> 상수 용매의 흐름 (1 ML / 분)와 압력 (1000 2000 PSI 사이의 특정 값)으로 HPLC 장치 (Spherisorb 열 연결되어 있음) 조정 및 튜브 매 1 분 변경 분획 수집기를 놓으십시오. </li><li> HPLC 장치에 샘플을로드하고 동시에 분획 수집기를 시작합니다. </li><li> 각 샘플에서 실행하고 표준 올리 고당 혼합물의 실행에서 1 ML 48 분수를 수집합니다. </li><li> 각 분획에서 섬광 약병 500 μl를 전송하고, 물 miscible 섬광 유체 3 ML과 내용을 섞는다. </li><li> 튜브를로드하고 신틸레이션 계수기에 참조하십시오. </li><li> 노출당 비용 (CPM) 판독이 분수 번호 기능으로 꾸몄다 있습니다. </li><li> 최대 노출당 비용 (CPM) 내의 값은 다음과 같이 특정 glycan 종의 대표 : 분수를 18-21도 M8, M9에 분수 36-39에 M5, M6 분수 22-26로, 분수를 27-31로 M7, 분수 32-35에 해당하는 분수 G2M9에 40-42로 G1M9과 분수 43-45. </li><li> 각 피크 내에서 분수에 대한 노출당 비용 (CPM) 값의 합계는 특정 glycan 종의 절대 금액을 반영합니다. 각 glycan 종의 절대 금액이 포함된 mannose 잔류물의 숫자로 나누어 자세한 mannose 잔류물을 포함하는 종 더 레이블, 다음과 같이 계산되는 각 glycan 종의 &quot;상대적 어금니 금액&quot;을 획득했다는 사실에 대해 보상하기 위해서는 . 이 값은 다음 각 특정 glycan 종에 대해 &quot;전체의 %를 &#39;얻기 위해 얻은 모든 glycan 수종의 상대 어금니 금액의 합계로 나눈 것입니다. </li></ol><table border="1"><tbody><tr><td style="text-align:center;"> 체이스 (H) </td><td style="text-align:center;"> 0 </td><td style="text-align:center;"> 4 </td></tr><tr><td> 엔도 H (CPM)과 함께 출시 </td><td> 90,000 </td><td> 16,800 </td></tr><tr><td> N - glycosidase F (CPM)와 B 출시 </td><td> 20,400 </td><td> 3900 </td></tr><tr><td> retentate (CPM) C에서 Glycoproteins </td><td> 371700 </td><td> 127695 </td></tr><tr><td> retentate (CPM) D에 Dolichol - oligosaccharides </td><td> 4350 </td><td> 540 </td></tr><tr><td> retentate에 Dolichol - oligosaccharides (%) E </td><td> 1.4 ± 0.7 </td><td> 0.2 ± 0.2 </td></tr></tbody></table> 표 1. deglycosidases과 당단백질 샘플에 dolichol - oligosaccharides의 존재의 N - 연결 설탕 체인 릴리스의 분석. 우리는 NIH 3T3 세포에서 lysate로 펄스 체이스 절차를 적용. Microcon 여과 후, 표시된 N - 연결 oligosaccharides는 펄스 라벨 (높은 mannose 타입) 후 복잡한 유형 (잠돌군 Zz - 처리 glycans)에 해당하는 것입니다 추격전이 기간이 지나면 N - glycosidase F 조로 추가 치료와 엔도 H 주로 출시되었습니다 엔도 H 저항 oligosaccharides. 이것은 정량 분석​​ dolichol - oligosaccharides가 초기 retentate에서 라벨의 사소한 분율 차지하는 사실을 확인했습니다. 참고 : <ol class="lalpha"><li> NIH 3T3 세포는 펄스는 [2-3 H]와 라벨이 붙지만 mannose하고 완전한 매체로 추격. Microcon YM30을 통해 세포 용해 및 여과 후, 높은 mannose oligosaccharides N - 연결은 엔도 H.와 부화하여 retentates에서 출시되었습니다 </li><li> (A)에서 Microcon의 retentates에서 엔도 H - 모성 자료는 다음 N - glycosidase F 조와 incubated 및 신틸레이션 계수기로 측정 oligosaccharides를 발표했다. </li><li> 메탄올 : 전구체 glycolipids를 제거하는 물 10시 10분 3초하고, 1N NaOH의 solubilized 및 섬광 카운터에서 계산 unextracted 소재 Microcon의 retentates는 (a) 엔도 H 처리하기 전에, 클로로포름과 4 번 추출되었다 같이 취득. </li><li> Dolichol - oligosaccharides은 (C)로 1에서 설명하고 섬광 카운터에서 계산에서 추출에서 정화했다. </li><li> retentate 총 노출당 비용 (CPM)의 dolichol - oligosaccharides (D) 비율 3 실험의 평균 (C + D). </li></ol> 대표 결과 <img src="/files/ftp_upload/1899/1899fig1.jpg" alt="그림 1" /> 그림 1. 치료 세포에서 proteasomal 억제에 총 NIH 3T3 glycoproteins의 펄스 체이스 분석 1 시간 후와 펄스 라벨. [2-3 H] 우리는 glucosylated과 예상 프로파일을 획득엽 성의 전구 물질은 (Glc 두 남자 9 GlcNAc 2 (G2M9)와 G1M9) 남아 있지만, M9에있는 라벨 및 M8 종의 대부분은, 후자는 모든 포도당의 트리밍 한 mannose 잔류물 (그림 1)의 결과를 모셨습니다. 아무도 무료로 mannose 또는 기타 작은 엽 성의 전구 물질은 정화의 철저에 증명, 발견되지 않았습니다. 다소 긴 8h 체이스 다음이 M7 - 6와 M5의 작은 금액 트리밍 더 광범위한했지만, 주요 수종은 M9와 M8 (그림 1 B)로 계속했다. 추적이 proteasomal 억제제 (30 μm의 MG - 132)의 존재로 이루어졌다면, 다음과 같은 손질 종의 사소한 축적 (그림 1 C)은 오직가 발생했습니다. <img src="/files/ftp_upload/1899/1899fig2.jpg" alt="그림 2" /> 그림 2. ERAD 기판의 설탕 체인의 정량 분석 총 glycoproteins에 비해. (A) 그림 1에있는 것과 유사한 실험에서, 각 올리 고당 종 상대 어금니 금액은 mannose 콘텐츠를 기반으로 계산했다. 우리는 각 glycan 종족에 대한 얻은 CPM 값을 변환, 모든 종류의 현재의 상대 어금니 금액의 총 합계를 기준으로 각 종족의 %가 후 두 실험의 평균 추적 시간의 함수로 꾸몄다되었습니다. (B)과 유사 을 (A) ERAD 기판 ASGPR H2a에서 공개 glycans를 제외하고 분석 나서 앞에서 4h 추적에 대한 proteasomal 억제제 MG - 132의 존재 또는 부재에 4h까지에 대한 펄스 상표와 체이스가. (C) 값 에서 MG - 132의 (A)와 (B) ASGPR H2a 및 당단백질 수영장에 대한 비교됩니다. 응급실에서 프로세스를 트리밍 N - 연결 설탕 체인 (D) 제도. 이들에 대한 M9 - 8에 비해 ERAD를 타겟으로하는 단백질 (R) 그 잠돌군 Zz와 근교로 종료합니다. 연결된 M6 - 5로 이어질 설탕 트리밍 처리 결과는 ERAD 프로세스 mannose 남은 5-6 mannose 잔류물과 종족을 항복하기 위해 N - glycans의 트리밍과 관련된 것으로 나타났습니다.

<ol>
	<li>Parodi, A. J., Behrens, N. H., Leloir, L. F., Carminatti, H. <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+polyprenol-bound+saccharides+as+intermediates+in+glycoprotein+synthesis+in+liver.">The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver.</a> Proc Natl Acad Sci U S A. 69 (11), 3268-3272 (1972).</li><li>Avezov, E., Frenkel, Z., Ehrlich, M., Herscovics, A., Lederkremer, G. Z. <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+(ER)+mannosidase+I+is+compartmentalized+and+required+for+N-glycan+trimming+to+Man5-6GlcNAc2+in+glycoprotein+ER-associated+degradation.">Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.</a> Mol Biol Cell. 19 (1), 216-225 (2008).</li><li>Frenkel, Z., Gregory, W., Kornfeld, S., Lederkremer, G. Z. <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-associated+degradation+of+mammalian+glycoproteins+involves+sugar+chain+trimming+to+Man6-5GlcNAc2.">Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.</a> J Biol Chem. 278 (36), 34119-34124 (2003).</li><li>Hosokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+endoplasmic+reticulum+(ER)+degradation+of+misfolded+Null+Hong+Kong+alpha1-antitrypsin+by+human+ER+mannosidase+I.">Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.</a> J Biol Chem. 278 (28), 26287-26294 (2003).</li><li>Jakob, C. A., Burda, P., Roth, J., Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+misfolded+endoplasmic+reticulum+glycoproteins+in+Saccharomyces+cerevisiae+is+determined+by+a+specific+oligosaccharide+structure.">Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.</a> J Cell Biol. 142 (5), 1223-1233 (1998).</li><li>Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+versus+secreted+forms+of+human+asialoglycoprotein+receptor+subunits.+Role+of+a+juxtamembrane+pentapeptide.">Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.</a> J Biol Chem. 271 (24), 14496-14503 (1996).</li></ol>

HPLC 분리 살 세포의 glycans의 펄스 체이스 분석 당단백질의 생활 전반에 걸쳐 올리 고당 구조 변경의 역학을 연구하는 방법을 제공합니다. 이러한 변경은 ER의 접이식, 품질 관리 및 인신 매매 시스템 2-5에 대한 신호를 생산에 관여하는 성장 증거가있다. 방법뿐만 아니라 관심의 특정 당단백질뿐만 아니라 우리가 대조 특정 ERAD 기판의 운명과 세포 당단백질 수영장의 비교이 문서에 설명된 ?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>600E Multisolvent delivery System controller</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT062710</td>
<td>&nbsp;</td>
<tr>
<td>Acetonitrile LiChrosolv (gradient grade for liquid chromatography)</td>
<td>&nbsp;</td>
<td>Merck Bioscience</td>
<td>1.0003</td>
<td>&nbsp;</td>
<tr>
<td>Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC503024 or 4208</td>
<td>&nbsp;</td>
<tr>
<td>Concentrator 5301, incl. 48 x 1.5 / 2.0 ml fixed-angle rotor</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5301 000.016</td>
<td>&nbsp;</td>
<tr>
<td>Dialyzed Foetal Calf Serum</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-011-1A</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modi&#xFB01;ed Eagle's Medium</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen Cell Culture</td>
<td>41965039</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Modi&#xFB01;ed Eagle's Medium - glucose free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D5030</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>EDTA disodium salt (Titriplex Iii)</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>1084180250</td>
<td>&nbsp;</td>
<tr>
<td>Endo Hf Kit (1,000,000 units/ml)</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>p0703S</td>
<td>&nbsp;</td>
<tr>
<td>Foetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-001-1A</td>
<td>&nbsp;</td>
<tr>
<td>Frac-100 fraction collector</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>18-1000-77</td>
<td>&nbsp;</td>
<tr>
<td>LS 6500 Liquid Scintillation Counting systems</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>510720</td>
<td>&nbsp;</td>
<tr>
<td>Mannose, D-[2-3H(N)]- (Specific Activity: 15-30Ci/mmol)</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>NET570A</td>
<td>&nbsp;</td>
<tr>
<td>N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>474790</td>
<td>&nbsp;</td>
<tr>
<td>N-glycosidase F (1000 units/ml)</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11365177</td>
<td>&nbsp;</td>
<tr>
<td>N-octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>O3757</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 cells</td>
<td>&nbsp;</td>
<td>American Type Culture Collection (ATCC)</td>
<td>CRL-1658</td>
<td>&nbsp;</td>
<tr>
<td>Opti-Flour</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>6013199</td>
<td>&nbsp;</td>
<tr>
<td>Phosphoric acid solution ( 49-51%, for HPLC)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>79607</td>
<td>&nbsp;</td>
<tr>
<td>Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2714</td>
<td>&nbsp;</td>
<tr>
<td>Protein A-Sepharose beads</td>
<td>&nbsp;</td>
<td>Repligen</td>
<td>IPA300</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit polyclonal anti-H2 carboxy-terminal 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>30970</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Bio-RAD</td>
<td>161-0301</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>342483</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution (100mM)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>Spherisorb NH2 Column, 5 &mu;m, 4.6 x 250 mm</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>PSS831115</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>BDH</td>
<td>306324N</td>
<td>&nbsp;</td>
<tr>
<td>Vibra-Cell ultrasonic processors VCX 750</td>
<td>&nbsp;</td>
<td>Sonics and Materials, Inc.</td>
<td>690-003</td>
<td>&nbsp;</td>
<tr>
<td>Buffer A: 1% Triton X-100, 0.5% w/v sodium deoxycholate, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer B: 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 stably expressing H2a 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer D: 0.5% Triton X-100, 0.25% w/v sodium deoxycholate, 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer E: 0.5% w/v SDS, 1% v/v 2-Mercaptoethanol, Protease inhibitor cocktail 2% v/v in PBS </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M3148</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

Glc의 첨부 파일<sub&gt; 3</sub&gt; 맨<sub&gt; 9</sub&gt; GlcNAc<sub&gt; 2</sub응급실에서 초기 polypeptides에 올리 고당&gt; 전구체는 분비 단백질에 대한 일반적인 수정입니다. 이 수정은 여러 생물 학적 과정에 연루되었지만, 그 기능의 추가 측면 당단백질 품질 제어 및 인신 매매에 대한 신호의 생산에의 역할 최근 증거로, 신흥 있습니다. 따라서, N - glycans 연결 및 처리에 관한 현상을 집중적으로 조사되고있다. 최근 proteomic 분석을 위해 개발되었습니다 방법은 크게 당단백질 N - glycans 연결의 특성을 향상했습니다. 그럼에도 불구하고, 그들은 관련 설탕 체인 처리의 역학에 대한 통찰력을 제공하지 않습니다. 이를 위해, 라벨링 및 펄스 - 체이스 분석 프로토콜은 일반적으로 복잡하며 매우 낮은 수율을주는 데 사용됩니다. 우리는 여기서 metabolically 표시된 N - 연결 oligosaccharides의 분리 및 분석을위한 간단한 방법을 설명합니다. 프로토콜은 [2 세포의 라벨을 기반으로 -<sup&gt; 3</sup&gt; H 관심 중 하나를 구체적으로 immunoprecipitated 단백질이나 분자 여과 (Amicon) 다음 엔도 H와 N - glycosidase F와 순차 요법에 의해 일반 당단백질 수영장에서] mannose, denaturing 용해와 oligosaccharides의 효소 놓습니다. 이 방법에서는 고립 oligosaccharides은 단일 monosaccharide의 해상도와 함께, 그들로 구성된 monosaccharide 단위의 숫자에 따라 다양한 glycan 구조 사이의 차별을 허용 HPLC 분석에 대한 입력으로 제공하고 있습니다. 이 방법을 사용하여 우리는 정상적인 조건에서 펄스 ​​체이스 후 proteasome 억제에 따라 총 세포 glycoproteins 높은 mannose N - 연결 올리 고당 프로파일을 공부할 수 있었다. 이 프로파일은 immunoprecipitated ER - 관련 저하 (ERAD) 기판에서 얻은 것과 비교했다. 우리의 결과는 대부분의 NIH 3T3 세포 glycoproteins은 비교적 안정되는 그들 oligosaccharides의 대부분이 인간의 손질하는 것이 좋습니다<sub&gt; 9-8</sub&gt; GlcNAc<sub&gt; 2</sub&gt;. 반면, 불안 정한 ERAD 기판은 인간을 손질 아르<sub&gt; 6-5</sub&gt; GlcNAc<sub&gt; 2</sub이 종 베어링&gt;을 glycoproteins은 proteasomal 저하의 억제에 축적.

Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

우리는 포유 동물 세포에서 생합성 후 glycoproteins의 초기 생활을 통해 N - 연결 glycans의 변조 분석을위한 방법을 설명합니다. 이것은 metabolically 표시 glycans, glycoproteins와 HPLC에 의해 시험에서 효소 릴리스의 펄스 체이스 분석에 의해 이루어진다.

포유 동물 세포에 Glycoproteins의 N - 연결 설탕 체인 펄스 체이스 분석

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

pulse-chase-analysis-n-linked-sugar-chains-from-glycoproteins

Research

JoVE Journal

Biology

15.1K 조회수.  Tel Aviv University. 우리는 포유 동물 세포에서 생합성 후 glycoproteins의 초기 생활을 통해 N - 연결 glycans의 변조 분석을위한 방법을 설명합니다. 이것은 metabolically 표시 glycans, glycoproteins와 HPLC에 의해 시험에서 효소 릴리스의 펄스 체이스 분석에 의해 이루어진다.

비디오: Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

자기편 및 비 점착 성의 포유 동물 세포의 현장 Subcellular 분별 (분리)에서

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

연구

생물학

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

포유 동물 세포의 DNA 이중 가닥 브레이크 (DSB) 수리 분석

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Microinjection하여 포유 동물 세포에서 mRNA 원자력 수출 속도론 분석

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

포유 동물 세포의 세포주기의 위치 분석

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

포유류의 링커 - 히스톤 Subtypes의 표현 분석

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

포유류의 세포에서 Endoplasmic 소포체 현지화 mRNAs의 시각화

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

포유 동물 세포에서 헴 합성 레벨 측정

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...