이 작품은 다카사키 대학 건강과 복지에 의해 지원 됩니다.

1. 성장 세포와 외 인 항 원 추가 biotin 단백질 라벨을 사용 하 여 <ol><li>준비 bOVA 키트 제조 업체에 따라 &#39; s 프로토콜. 
	참고: 일반적으로, bOVA 포함 2 M biotin 1 M OVA 당 평균.</li>
	<li>성장 DC2.4 셀 RPMI-1640 2mm L-글루타민, 1mm 나트륨 pyruvate, 0.1 m m 불필요 한 아미노산, 100 U/mL 페니실린-스, 55 m m 2-mercaptoethanol, 10 mM HEPES (pH 7.5), 및 5에서 37 ° C에서 10% 태아 종 아리 혈 청 (여기서 부터는 RPMI)와 보충 습도 인큐베이터에서 % CO 2 (이 언급 없이 세포는 인 큐베이 팅이 조건에서). 그것은 또한 DMEM 10% 태아 종 아리 혈 청 RPMI 대신 NaHCO 3 (여기서 부터는 DMEM) 3.7 g/L 보충을 사용 하 여.</li>
	<li>주 정화, 전에 분할 RPMI의 셀 사용 하 여 조직 문화 접시에 1 x 10 5 셀/ml. Confluent 상태에서 세포를 유지 하지 마십시오. DC2.4 셀 매우 반 confluent 상태로까지 외 인 항 원을 통합할 수 있지만 빠르게 오버 합칠 상태에 도달 후 능력을 잃고.</li>
	<li>DC2.4 전에 세포는 반 confluent 1 x 10 6 셀/ml bOVA의 250 µ g/mL을 추가 하 고 2-4 헤에 대 한 품 어 
	참고: 다운스트림 실험 동안 모든 버퍼 및 시 약 보관 4 ° C에서 달리 명시 하지 않는 한.</li>
</ol> 2. Microsomes의 준비 <ol><li>새 50 mL 원뿔 관으로 조직 접시에서 부드러운 pipetting으로 DC2.4 세포를 수확.</li>
	<li>5 분, 4 ° C 1000 x g에서 원심 분리기와 포부에 의해 bOVA와 매체를 조심 스럽게 제거.</li>
	<li>DC2.4 셀 1000 x g 5 분, 4 ° C에서 원심 분리 하 여 PBS 버퍼 (1.37 m m NaCl, 8.1 m 나 2 HPO 4, 2.68 m KCl, 1.47 m m KH 2 포 4 m m)의 40 mL로 두 번 세척 하 고 상쾌한 포부에 의해 때마다 삭제.</li>
	<li>Resuspend 단계 2.3에서 1-2에서에서 펠 릿 mL (문화 매체의 1/20-1/10 볼륨) 프로 테아 제 억제 물 칵테일 및 전송 resuspended DC2.4의 1/1000 양으로 균질 매체 (0.25 M 자당, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4])의 세포에 얼음 처럼 차가운 Dounce 균질 화기.</li>
	<li>차가운 Dounce 균질 화기와 10-20 스트로크에 의해 resuspended DC2.4 세포를 부드럽게 중단. 
	참고: 중단 단계 동안 Dounce 균질 화기는 냉각 얼음에</li>
	<li>새 15 mL 원뿔 튜브 셀 중단 중지 전송.</li>
	<li>10 ml 총 8-9 mL 균질 매체를 추가 하 고 원뿔 튜브 2000 g x 4에서 10 분 원심 ° c.</li>
	<li>새 15 mL 원뿔 튜브 손상 되지 않은 세포와 핵, 제거 하는 상쾌한 전송 및 2000 g x 4에서 10 분 동안 다시 원뿔 튜브 원심 ° c.</li>
	<li>새로운 ultracentrifugation 튜브와 g x 100000 및 4에서 45 분 동안 원심 분리기에는 상쾌한 전송 ° c.</li>
	<li>는 상쾌한 발음 하 고 다운스트림 실험 microsome 분수를 만들기 위해 여러 번 위아래 솔루션 pipetting으로 펠 릿 프로 테아 제 억제 물 칵테일의 1/1000 양으로 균질 매체의 1-2 mL에 신중 하 게 resuspend.</li>
	<li>새 5 mL 둥근 바닥 관으로 microsome 분수 전송. 
	참고:이 단계 후에 그것은 제조 업체에 따르면 iodixanol 밀도 그라데이션 원심 분리에 의해 객관적인 microsomes을 풍부 하 게 &#39; s 프로토콜, 일반적인 microsomes를 제거 하려면. 외 인 항 원에 대 한 피크 분수 및 수집한 다음 정화 (3 단계)의 다음 단계로 들어서는.</li>
</ol> 3. BOVA와 Microsomes의 정화를 받고 ERAD microsome 분수의 단계 2.11 5 mL <ol><li>신선한의 추가 1/100 볼륨 SA 자석 구슬 원형 하단 튜브. 
	참고:이 단계를 하기 전에 그것은 미리 microsome 분수를 줄이기 위해 일반적인 microsomes의 오염 제어 자석 구슬에 의해 취소.</li>
	<li>부드럽게 잘 혼합 하 고 4 시 30 분 동안 천천히 라운드 하단 튜브를 회전 ° c.</li>
	<li>라운드 하단 튜브에 총 4 mL에 균질 매체의 2-3 mL를 추가 하 고 부드럽게 잘 섞어.</li>
	<li>라운드 하단 튜브 마그네틱 스탠드에 놓고 4 ° c.에서 10 분 동안 품 어 이후는 소포에 바인딩된 구슬 자석에 끌리는, 갈색 마이크로 구슬 점차적으로 자석에 가장 가까운 튜브 벽에 축적 됩니다.</li>
	<li>튜브와 함께 신중 하 게 언바운드 소포를 제거 하는 열망에 의해 여 supernatants를 버리고 마그네틱 스탠드에 남아.</li>
	<li>자석 구슬 자석에 의해 균질 매체의 5 mL로 두 번 소포에 바인딩된 워시 4 ° C에서 10 분 서 하 고 신중 하 게 열망 하 여는 상쾌한 때마다 삭제. 
	참고: 마그네틱 스탠드, 10 분, 2000 x g에서 centrifugations에 의해 정화 없이 4 ° C는 또한 사용할 수.</li>
	<li>자석 구슬 몇 번 위아래로 솔루션 pipetting 100 µ L 균질 매체에 신중 하 게 소포를 준수할 resuspend.</li>
	<li>다운스트림 실험에 대 한 정화 microsome로 새로운 microtube에 resuspended 소포 전송. 
	참고: 일반적으로, 1 x 10 7 셀에서 5-20 µ g 단백질을 포함 하는 50 µ L microsome 분수 고립 될 수 있다.</li>
</ol> 4. 순화 Microsomes의 분석 <ol><li>Resuspend 자석 구슬에서 소포에 바인딩된 TNE 버퍼의 100-200 µ L 3.8 단계 (20 mM Tris HCl pH 7.4, 150 m m NaCl, 0.5 M EDTA, 1% Nonidet P-40) 프로 테아 제 억제 물 칵테일의 1/1000 볼륨 균질 매체 대신.</li>
	<li>는 lysate에서 전송 단계 4.1 새로운 1.5 mL microtube.</li>
	<li>제조 업체 당 BCA 분석 결과 키트를 사용 하 여 단계 4.2의 단백질 농도 결정 &#39; s 프로토콜. 
	참고: 일반적으로 5-10 µ L 단계 4.2에서에서 lysate 단백질 농도 결정 하기 위해 충분 하다.</li>
	<li>는 lysate에서 전송할 단계 (실버 얼룩에 대 한 단백질의 2 µ g 및 서쪽에 게 더 럽 히기를 위한 단백질의 10 µ g) 4.2 새로운 microtubes.</li>
	<li>SDS 겔-로딩 버퍼 x 1에서 95 ° C와 종 단백질에서 열 블록에 있는 microtubes를 넣어 (100 mM Tris HCl pH 6.8, 200 m m dithiothreitol, 4 %SDS, 0.2 %bromophenol 블루, 20% 글리세롤) 5 분에 대 한</li>
	<li>21500 g x 4에서 10 분 동안 microtubes 원심 ° C. 불용 성 부분을 제거 하는 새로운 microtubes에 있는 supernatants 수집.</li>
	<li>단계 4.6 (2 µ g) 표준 SD-페이지에서 해결된 단백질 분석.</li>
	<li>시각화 실버 얼룩은 얼룩을 사용 하 여 단백질 밴드 키트 제조 업체에 따라 &#39; s 프로토콜.</li>
	<li>표준 SD-페이지와 서 부 럽 4.6 (10 µ g) 단계에서 확인 된 단백질 분석. 시 약 (SA-HRP)를 사용 하 여 제조 업체 당 &#39; s 프로토콜 및 fluorography에 의해 시각화.</li>
</ol> 5. 생체 외에서 BOVA 사용 하 여 정화 Microsomes의 ERAD Ubiquitination의 재구성 단계 3.8 reticulocyte lysate (RL)의 50% 볼륨으로 반응 버퍼 (50 mM Tris pH 7.4, 3mm ATP, 0.5 m m MgCl x 1에서에서 <ol><li>이전에 순화 microsomes (5-10 µ g 단백질) 2), 그리고 새로운 microtube에 플래그 태그 유비퀴틴 오후 0 시 2 분. 이 실험의 최종 볼륨은 20-40 µ L.</li>
	<li>품 37에서 2 h microtube ° C. 
	참고: 단계 5 월 12 일에서에서 ubiquitinated bOVA 양의 서쪽 blotting에 의해 감지 너무 작으면, 추가 10 µ M의 MG132 또는 proteasomes에 의해 ubiquitinated bOVA의 처리를 억제 하는 lactacystin의 2 µ M.</li>
	<li>얼음에 microtube 배치 하 여 반응을 중지</li>
	<li>는 microsomes 프로 테아 제 억제 물 칵테일의 1/1000 양으로 500 µ L TNE 버퍼를 추가 하 여 해결.</li>
	<li>21,50에서 10 분 원심 분리기는 microtubeg와 4 x는 0 ° c.에 생체 외에서 ubiquitination 분석 결과 전에 DC2.4에서 ubiquitinated bOVA 포함 하는 불용 성 부분을 제거 하는 새로운 microtube는 supernatants 수집.</li>
	<li>는 상쾌한 새로운 microtube 전송과 새로운 SA 자석 구슬 (일반적으로 5 µ L supernatants의 500 µ L에 대 한)의 1/100 볼륨 추가.</li>
	<li>부드럽게 잘 혼합 하 고 30 분 동안 천천히 microtube 4 회전 ° c.</li>
	<li>Microtube 21500 g x 4에서 10 분 원심 ° C. 삭제 bOVA 및 ubiquitinated bOVA 열망 하 여 상쾌한 바운드 SA 자석 구슬.</li>
	<li>TNE 버퍼의 1 mL와 함께 두 번 수집된 사 자석 구슬 21500 g x 4에서 10 분 동안 원심 분리 하 여 세척 ° C. 삭제 상쾌한 포부에 의해 각 시간.</li>
	<li>SA 자성 구슬에 의해 순화 된 단백질을 해결 하기 위해 열 블록에 95 ° C에서 5 분에 대 한 버퍼를 로드 하는 SDS 젤 x 1에 SA 자석 구슬 종.</li>
	<li>Microtube 21500 g x 4에서 10 분 원심 ° C에 불용 성 부분을 제거 하는 새로운 microtube는 supernatants 수집.</li>
	<li>분석 표준 SD-페이지 및 서쪽 더 럽 히는 단백질에 준수할 SA 자석 구슬. (안티, 안티-멀티-Ub, 깃발과 반대로 마우스 IgG HRP) 항 체 및 시 약 (SA-HRP)의 사용은 제조 업체 당 &#39; s 프로토콜 및 fluorography에 의해 시각.</li>
</ol>

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저자는 공개 없다.

CP의 이전 연구에서 통합된 외 인 항 원 immunofluorescent 현미경 검사 법16,30,,3132에 의해 늦은 endosome 또는 응급실의 제한 된 영역에서 축적 된. 세포질 구획은 자당 또는 iodixanol 조밀도 기온 변화도 원심 분리를 사용 하 여 식별 되는 ERAD와 같은 전송 및 외 인 항 원 처리 수행 됩니다 응급실 또는 늦은 endoso...

MHC I 분자 cytosol1에서 유비퀴틴-프로테아좀 시스템에 의해 처리 되는 내 인 성 항에서 파생 하는 짧은 항 원 펩 티 드와 모든 nucleated 세포의 표면에 표현 된다. 처리 후, 항 원 펩 티 드 펩 티 드 운송업 자 탭에 의해 바인딩과 그물 (응급실) 루멘으로 이송 됩니다. ER의 루멘에서 일련의 특정 보호자 펩 티 드 로드 하 고 올바른 접는 MHC의 나 복잡 한 지원 합니다. 분자의이 시리즈는 펩 티 드 로드 복잡 한 (PLC), 응급실 펩 티 드 나2MHC에 적재에 대 한 중앙 구획을 나타내는 이라고 합니다. 후에 펩 티 드, 로드는 MHC I 분자 세포 표면에 수송 하 고 핵심적인 역할을 담당할 적응 면역 시스템에 자기 마커, 그리고 수는 CD8+ 세포 독성 T 세포 (Ctl) 암 세포 또는 전염 성 요원을 항 원에 의해 3비 각자 단백질 펩 티 드.
항 원 제시 세포 (APC), 외 인 항에서 항 원 펩 티 드에서는 또한 내가4,5,6,,78 통해 CP, 주로 수행 MHC 시 제시 의해 Dc9,,1011. CP는 필수+ T 순진한 CD8의 활성화를 위한 둘 다 세포와 CD8 메모리+ T 세포의 비활성화에 의해 안티 전염 성 및 안티-종양 Ctl12,13, 그리고 면역 관용의 유지 보수에 자기 행동 naïve T 세포14,15. 그러나 CP CP의 분자 기계 장치는 아직 자세히 설명 될 적합 한 면역 계통에 많은 중요 한 역할을 재생 합니다. CP의 이전 연구 밝혔다 exogenous 항 모두 응급실에는 endosome 현지 했다 ERAD, ERAD 같은 처리 및 펩 티 드16 로드에 대 한 응급실에 외 인 항 원은 endosome에서 이송은 제안 처리 . 그러나, 축적 증거가 나타냅니다 응급실에서 아닙니다 그러나 오히려 응급실 (그림 1)17,18의 독특한 기능을가지고 아닌 클래식 endocytic 구획 밖으로 CP의 펩 티 드 로드 수행 ,19,,2021. Aminopeptidase의 높은 활동에 의해 항 원 펩 티 드 선구자의 저하를 방지 하려면22 cytosol, 처리 및 CP에서 로드 하는 펩 티 드에이 아닌 클래식 endocytic 구획 (그림 1)의 인접 지역에서 발생 합니다. 이러한 endocytic 구획의 characterizations 논란이 있지만, 이외에 외 인 항 원이이 구획에 없는 기존 특정 분자 있다.
ERAD는 세포질 통로, 특히 응급실에서 misfolded 단백질을 제거 하는. ERAD 경로에 misfolded 단백질 retrogradely ER 막 세포질을 통해 운반 되며 유비퀴틴-프로테아좀 시스템23,,2425처리. 큰 분자, 단백질, 등 지질 bilayer를 통해 수송 된다,이 분자를 통과에 복잡 한 복잡 한 Sec61 및 Derlin ER26, 그리고 복잡 한 톰에 복잡 한 팀 같은 translocon 라는 분자 장치는 미토 콘 드리 아27 것 추가 항 ER 막 통해 수송 된다, 그들은 translocons, Sec61 복합 등으로 복잡 한 지질 bilayer를 관통 하 게 해야 합니다. 여기 설명 하는 방법을 endocytic 구획에 대 한 표식으로이 막 관통 분자를 이용 하 여 타겟된 기를 정화.
여기에 설명 된 방법은 외 인 항 원으로는 DC 같은 셀 선 DC2.428 와 biotinylated ovalbumin (bOVA)를 사용 하 여 새로운 기 정화 프로토콜입니다. Endocytic 구획 streptavidin (SA)에 의해 순화 되었다-자석 구슬 메이커로 막 관통 bOVA를 사용 하 여. 이 순화 microsome 일부 것 추가 bOVA 여전히 막 분수에 보존 되었다 하지만 microsome, 및 다음 ubiquitinated의 외부에 이송 되었고 처리29 생체 외에서. 이 순화 microsome endocytic 구획 특정 단백질 뿐만 아니라 ER-상주 단백질 ERAD 및 펩 티 드 복잡 한; 로드 포함 세포질 구획 CP29에 대 한 예비 endocytic 구획 제안. 이 프로토콜은 exogenous 항 원의 종류에 따라 그리고 다른 DC 하위 집합 및 다른 세포 유형, 대 식 세포, B 세포, 내 피 세포, 등 실력 CP에 대 한 Dc의 정확한 분자 메커니즘을 명확 하 게 적용 됩니다.

<table border="0" cellpadding="0" cellspacing="0" width="871">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RPMI 1640</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11875-093</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fetal bovine serum</td>
			<td style="width:239px;">Equitech bio</td>
			<td style="width:119px;">SFB30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sodium pyruvate</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11360-070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:272px;">MEM non-essential amino acids</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">HEPES</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">2-mercaptoethanol</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">L-glutamine</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">25030-164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Penisicillin-Sreptomycin</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">12100-46</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OVA</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Biotin-protein labelling kit</td>
			<td style="width:239px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F6347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MG-132&#160;</td>
			<td style="width:239px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">201270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">lactacystin&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">L6785</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Dounce homogenizer</td>
			<td style="width:239px;">IUCHI</td>
			<td style="width:119px;">131703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">protease inhibitor cocktails&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:272px;">iodixanol&#160;</td>
			<td style="width:239px;">Cosmo bio</td>
			<td style="width:119px;">1114542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">SA-magnetic beads&#160;</td>
			<td style="width:239px;">New England Biolabs</td>
			<td style="width:119px;">201270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">control magnetic beads</td>
			<td style="width:239px;">Chemagen</td>
			<td style="width:119px;">M-PVA012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">magnetic stand</td>
			<td style="width:239px;">BD Biosciences</td>
			<td style="width:119px;">552311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BCA protein assay kit</td>
			<td style="width:239px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">silver staining kits</td>
			<td style="width:239px;">Cosmo bio</td>
			<td style="width:119px;">423413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Reticulocyte Lysate</td>
			<td style="width:239px;">Promega</td>
			<td style="width:119px;">1730714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Flag-tagged ubiquitin&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">U5382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-ovalbumin (OVA,mouse)</td>
			<td style="width:239px;">Antibody Shop</td>
			<td style="width:119px;">HYB 094-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">ant-multi-ubiquitin (mouse)</td>
			<td style="width:239px;">MBL</td>
			<td style="width:119px;">D058&#8722;3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-Flag (mouse)</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">trypsin</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">85450C</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

수지상 세포 (Dc)는 고도의 처리 및 중요 한 조직 적합성 클래스 (MHC)에 따라 내 면된 외 인 항 원 제시의 나 분자 라고 크로스-프레 젠 테이 션 (CP). CP는 중요 한 역할 뿐만 아니라 순진한 CD8 자극+ T 세포와 CD8 메모리+ T 세포에 대 한 감염 및 self-acting의 비활성화에 뿐만 아니라 종양 면역 naïve T 세포 T 세포 아 또는 T 셀 삭제. CP의 중요 한 분자 메커니즘 해명 될 남아, 비록 축적 증거가 나타냅니다 exogenous 항 저하를 통해 떠다니고 그물 관련 (ERAD) 비 클래식에서 수출 후 endocytic 처리 됩니다. 구획입니다. 최근까지, 외 인 항 원 이외의 아무 특정 분자 마커 있었기 때문에 이러한 endocytic 구획의 characterizations을 제한 했다. 여기에 설명 된 메서드는 이러한 endocytic 구획의 정화에 대 한 수 있는 새로운 소포 격리 프로토콜입니다. 이 순화 microsome 사용 하 여, 우리 재구성 ERAD 같은 전송, ubiquitination, 및 처리는 외 인 항 원에 체 외에, 유비퀴틴-프로테아좀 시스템 수출이 후 외 인 항 원 처리 제안 세포질 구획입니다. 이 프로토콜은 CP의 분자 메커니즘을 명확 하 게 다른 세포 유형에 더 적용할 수 있습니다.

CP의 분자 메커니즘을 명료 하 게 그것은 exogenous 항 ERAD와 같은 전송 및 처리를 받아야 세포질 구획을 식별 하는 데 필요한. Immunofluorescent 현미경 또는 전자 현미경 관찰 어디 외 인 항 원16,,1718,19, 축적 된 세포질 구획을 확인 하는 동안 3...

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

여기에 설명 된 메서드는 exogenous 항 바인딩과 그물 관련 저하 크로스-프레 젠 테이 션에 의해 처리 됩니다 세포질 구획의 정화에 대 한 수 있는 새로운 소포 격리 프로토콜입니다.

외 인 항 원 십자가-프레 젠 테이 션에의 바인딩과 그물 관련 성능 저하 막 구획의 정화

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

purification-membrane-compartment-for-endoplasmic-reticulum

Research

JoVE Journal

Immunology and Infection

8.2K 조회수.  Takasaki University of Health and Welfare. 여기에 설명 된 메서드는 exogenous 항 바인딩과 그물 관련 저하 크로스-프레 젠 테이 션에 의해 처리 됩니다 세포질 구획의 정화에 대 한 수 있는 새로운 소포 격리 프로토콜입니다.

비디오: Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipid 항원의 대량 Spectrometric 분석

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

연구

면역학 및 감염병학

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

막 극성, 멤브레인 무결성 및 세포 내 이온 농도의 모니터링 변경<em&gt; 폐렴 구균</em&gt; 형광 염료를 사용

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA 정제는 세포 내에서 재배<em&gt; 리스테리아</em&gt; 식세포 세포에

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

X 선 결정학, 생물 물리학 및 기능 분석 실험을 사용하면 메커니즘 집행 T 세포 암 항원의 수용체 인식을 확인하는 방법

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

외 인 항 원 비 유전 표면 장식 Avidin 비타민 b 복합체 시스템을 통해 진 균 bovis BCG 백신에의 표현

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

해 다중 단백질 신호 복합물 분자 보완성 친 화력 정화 (BiCAP)에 의해

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

조립 및 프로토 타입 거품 바이러스 Intasomes의 정화

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

외 인 항 원 십자가-프레 젠 테이 션에의 바인딩과 그물 관련 성능 저하 막 구획의 정화

요약

더 많은 비디오 탐색

이 비디오의 챕터

Glycosphingolipid 항원의 대량 Spectrometric 분석

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

막 극성, 멤브레인 무결성 및 세포 내 이온 농도의 모니터링 변경

RNA 정제는 세포 내에서 재배

X 선 결정학, 생물 물리학 및 기능 분석 실험을 사용하면 메커니즘 집행 T 세포 암 항원의 수용체 인식을 확인하는 방법

관련된 바이러스 및 세포질 단백질의 Id에 대 한 바이러스 성 DNA의 정화

외 인 항 원 비 유전 표면 장식 Avidin 비타민 b 복합체 시스템을 통해 진 균 bovis BCG 백신에의 표현

인간의 과발현과 정제

해 다중 단백질 신호 복합물 분자 보완성 친 화력 정화 (BiCAP)에 의해

조립 및 프로토 타입 거품 바이러스 Intasomes의 정화