이 연구는 SH에 도이치 Forschungsgemeinschaft GraKo1121, Hu1011/2-1 및 SFB940의 교부금에 의해 지원되었다

참고 : 프로토콜에 표시된 버퍼에 대한 자세한 정보는 표 1에서 사용할 수 있습니다. 1. 포름 알데히드는 살아있는 세포 간 연결하여 단백질 - 단백질 상호 작용의 고정 <ol><li> 성장 미디어, 주식 솔루션 및 버퍼 P1을 준비 시작하려면 <ol><li> 500 ㎖의 배지를 준비 매체 막 미끼 단백질과 먹이 단백질 간의 상호 작용을 지원하는 조건을 제공한다. 또, 하나의 실험에서는 어떠한 상호 작용 (가교없이 예) 예상되는 조건 하에서 수행되어야한다. 참고 : 우리는 스트레스가 아닌 스트레스를 박테리아의 단백질 - 단백질 상호 작용을 비교합니다. 따라서, 우리는 우리가 2 L 삼각 플라스크에 스트레스 유도제 (예를 들어, 0.5 M의 NaCl)으로 보완 500 ㎖의 루리아 베르 타니 (LB) 배지 (표 1)를 준비하고 있습니다. 제어를 위해, 우리는 0.17 M 염화나트륨 (산도 7.0)와 일반 LB 매체를 사용합니다. </li><li> 트리스 버퍼를 준비합니다 (20 mM 트리스-HCL;의 pH 8.0) 및 0.1 M 에틸렌 디아민 테트라 아세트산 (EDTA), pH를 8.0 원액 (표 1). </li><li> 버퍼 P1을 준비합니다. 트리스 완충액에서 0.5 M의 최종 농도에 자당을 용해. 여과에 의해 버퍼 P1을 소독하고 4 ℃에서 저장 </li></ol></li><li> 벡터에서 C-말단 패 혈성 태그 융합으로 막 미끼 단백질을 발현하는 세포를 성장. 충분한 시간 동안 멤브레인 미끼 단백질의 발현을 유도한다. 참고 : 우리는 500 ml의 매체에 250 ㎕의 1 M 이소 프로필-β-D-티오 갈 락토 피 라노 시드 (IPTG)를 첨가하여 초기 로그 단계 (A 600 = 0.3)에서 (최종 농도 : 0.5 mm)를 세균의 막 미끼 단백질의 발현을 유도 . 충분히 식을 수 있도록하기 위해, 우리는 늦은 로그 단계 (A 600 = 1.2) 때까지 박테리아를 배양한다. </li><li> 300 ㎖의 각각의 최소 볼륨 각 문화 두 개의 원심 분리기 비커를 준비하고 얼음에 배치합니다. </li><li> 포름 알데히드 가교를 수행합니다. CAUTION : 포름 알데히드는 독성이 강한 우리가 안전 흄 후드에서 작업하는 것이 좋습니다.. <ol><li> 안전 흄 후드에서 배양 용기를 전송합니다. (- X)와 가교 포름 알데히드 (+ X)를 포함하여 한 두 개의 샘플, 가교를 생략 하나에 각각의 샘플을 분할합니다. 0.6 %의 최종 농도에 도달하기 위해 250 ㎖의 배양을 4 ml의 37 % 포름 알데히드 용액을 추가. </li></ol></li><li> 다시 문화 혈관을 전송하고 이전과 추가 20 분 동안 세포를 성장. </li><li> 안전 흄 후드 아래에 원심 분리기 튜브로 문화를 기입하십시오. 30 분 3,000 XG에서 원심 분리하여 세포를 수집합니다. </li><li> 신중하게 안전 연기 후드 아래에 그것을 피펫으로 상층 액을 버린다. 독성 포름 알데히드 함유 상층 액을 수집하고 적절하게 처리하십시오. </li><li> 막 단백질의 준비 기간 동안 최적의 수율에 도달하기 위해 먼저 spheroplasts를 준비합니다. 여기, 우리는 그람 음성 박테리아의 스페 형성을위한 프로토콜을 제공 : <ol><li> Prepar전자 버퍼 P2 (표 1) 동결 건조 분말. 부드럽게 1.5 ML 튜브에, 0.1 M EDTA, pH가 8에서 2 밀리그램의 라이소자임을 녹입니다. 항상 실험의 하루 갓 버퍼 P2를 준비합니다. </li><li> 단백질 분해 효소 억제제 (버퍼 P3)과 트리스 버퍼를 준비합니다. 트리스 완충액 10 ㎖로, 10 mM의 최종 농도로 0.1 ㎖의 1 M 페닐 메틸 (PMSF) (표 1)을 추가한다. 프로테아제 억제제 PMSF의 기능이 제대로 동작하는지 확인하기 위해 즉시 버퍼를 사용합니다. </li><li> 단백질 분해 효소 억제제와 15 ML 원뿔 튜브로 전송 솔루션 10 ㎖ 트리스 버퍼를 Resuspend 세포 펠렛. </li><li> 1 ㎖ 버퍼 P2를 추가하고 30 분 동안 얼음에 품어. </li><li> 30 분 3,000 XG에서 원심 분리하여 spheroplasts를 수집하고 조심스럽게 상층 액을 버린다. </li></ol></li><li> -20 ℃에서 하룻밤 스페 펠렛을 품어 </li></ol> 2. 패 혈성 태그의 막 단백질의 정제 (미끼) <ol><li> 얼음에 스페 펠렛을 놓습니다. &lt;/ 리&gt; <li> 스페 펠렛은 얼음에 해동 시간 동안 각 스페 준비를 위해 10 ㎖ 버퍼 P3를 준비합니다. 조심스럽게 10 ML 트리스 버퍼에 1 mg의 DNaseI을 녹입니다. 사용 직전에 0.1 ㎖의 1 M의 PMSF를 추가합니다. </li><li> 6 ㎖를 새로 제조 P3에서 펠렛을 재현 탁. spheroplasts을 방해하기 위해, 각 버스트 사이에 1 분의 일시 정지와 얼음에 지속적으로 1 분 동안 샘플 네 번 초음파 처리. </li><li> 수확 된 세포 파편을 10 분 동안 10,000 XG에서 샘플을 원심 분리기. 초원 심 분리기 튜브에 피펫을 사용하여 상층 액을 전송합니다. </li><li> 30 분 동안 100,000 XG에서 막 분획을 펠렛. 를 용해하지 않고 트리스 버퍼에 조심스럽게 펠렛을 씻으십시오. 종이 조직 (예를 들면 휴지)와 튜브를 건조. 언제든지 펠​​렛을 방해 피한다. </li><li> 1 ㎖ 트리스 버퍼에 조심스럽게 펠렛 (= 세포막을)를 Resuspend. 예 BCA 분석하여 단백질 농도를 결정하기 위해 20 μL 나누어지는을 사용합니다. 이 단계에서, 멤브레인은 충격에 리 얼 수pond 지폐 질소 -80 ° C.에 저장 </li></ol> 3. 패 혈성 태그 막 미끼 단백질 및 SDS-PAGE의 정화 <ol><li> 트리스 완충액으로 5 ㎎ / ㎖로 막 분획의 단백질 농도를 정상화. 초 원심 분리기 튜브에 2.5 ml의 막 분획 (5 ㎎ / ㎖)를 취하여 막 단백질을 가용화하기 위해, 2 %의 최종 농도에 도달하기 위해 20 % 트리톤 X-100의 0.25 mL를 넣고. 마이크로 자석 막대를 추가하고 1 시간 동안 얼음에 저어. 참고 : 일반적으로 우리는 세제로 트리톤 X-100을 사용하여 좋은 결과를 가지고 있지만, 그것은 최적화를위한 세제를 변경하는 것이 유용 할 수 있습니다. 이러한 관점에서, 우리는 각각의 멤브레인 미끼 단백질의 기능적 정제에 사용되는 세제 최상의 결과가있다. </li><li> 3.1 단계를 수행하는 동안, 50 ㎖는 (표 1)에서 W와 5 ㎖ 버퍼 E 버퍼 준비합니다. 10 ㎖ 5 배 버퍼 W 농축 ML 50을 입력하고 150 ㎕의 20 % 트리톤 (Triton) X-100을 추가합니다. <ol><li> 1 ㎖ 배 buffe를 기입R의 E의 10 ㎖로 농축하고 30 ㎕의 20 % 트리톤 (Triton) X-100을 추가합니다. 8 ㎖의 버퍼 W.와 1 ㎖ 패 혈성-Tactin superflow 중력 흐름 열 평형 주 : 정제 절차 중에 어떤 버퍼 내의 임계 미셀 농도 (CMC) 이상의 농도의 10 배에서의 가용화에 사용되는 세제를 추가하는 것이 필수적이다. </li></ol></li><li> 불용성 막 분획을 펠렛 30 분 동안 100,000 XG에서 가용화 샘플 및 초 원심 분리기에서 마이크로 자기 막대를 제거한다. 추가의 정제를위한 피펫을 사용하여 상층 액을 제거합니다. </li><li> 열을 뜨는을로드합니다. 중력의 흐름에 열을 실행합니다. 이 세척 단계 배를 반복하여 5 ㎖ 버퍼 W.에 열을 씻으십시오. </li><li> 이 용출 단계 반복 배속 1 ㎖ 버퍼 E.으로 막 미끼 단백질을 용출. </li><li> 원심 분리 필터 장치로 용출 분획 2, 3 및 4-300 μl를 집중한다. </li><li> 50 ㎕의 5 배 SDS-PAG 각 샘플의 200 μl를 혼합E 로딩 염료. 125 μL 씩 분주 각 준비를 분할합니다. 포름 알데히드 상호 링크를 반대하는, 95 ° C에서 20 분 동안 각각 준비 하나 나누어지는 끓인다. 샘플 벤치 위에 적어도 10 분 동안 실온으로 냉각 할 수 있습니다. </li><li> 면역 블롯에 적합한 폴리 아크릴 아마이드 겔의 하나의 레인에로드 각 시료 30 μl를. 가장 좋은 방향으로의 prestained 분자량 마커를 사용하여 SDS-PAGE (10)을 실행합니다. </li></ol> 4. 면역 블롯 분석 상호 작용 파트너를 확인하려면 <ol><li> 일단 7.6이 예 반 건조하여 니트로 셀룰로오스 막에 전사 단백질을 완료 단계. </li><li> 비특이적 라벨링을 방지하는 멤브레인을 차단. 0.05 % 트윈 -20 (TBS-T)의 최종 농도로 보충 트리스 - 완충 식염수 부피 당 3 중량 %를 달성하기 위해 소 혈청 알부민 (BSA)을 계량하여 차단 완충액 준비한다. 멤브레인을 포함하는 버퍼를 차단하는 충분한 양을 사용합니다. 실온에서 1 시간 동안 멤브레인을 차단합니다. </li><li> 희석ND 평소와 같이 먹이 단백질 특정 첫 번째 항체를 배양한다. 두 번째 항체로 HRP 결합 된 항체를 사용합니다. </li><li> 고감도 화학 발광 검출 키트를 사용하여 면역 블롯을 개발한다. 고전 막 처리 절차 또는 디지털 촬영 장비를 사용하여 신호를 모니터한다. </li></ol> 5. 상호 작용 파트너를 식별하는 NanoLC-ESI-MS/MS 높은 해상도 실험 <ol><li> 확인되어야한다 특정 항체가 먹이 단백질 또는 알 수없는 상호 작용 파트너를 사용할 수없는 경우, 식별을위한 질량 분석 (MS)를 사용합니다. 각질 오염을 방지하기 위해서, 단백질 분리를위한 precasted 겔을 사용한다. </li><li> 실버는 제조자의 프로토콜에 따라 MS 호환 염색 키트를 사용하여 SDS-PAGE 얼룩. 유리 탱크에있는 염색 및 세척 단계를 수행합니다. </li><li> 소비세 각 밴드와는 고해상도 LC / MS (9)에 의해 이들을 분석한다. </li></ol>

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우리는 공개 아무것도 없어.

막 척추 분석을 확인하고 막 단백질의이 시점 알 수없는 상호 작용 파트너 식별을 가능하게하는 생화학 적 방법입니다. 막 척추 연쇄상 구균 태그가 막 미끼 단백질의 정제와 포름 알데히드에 의해 생체 내 가교에 결합한다. 면역 블로와의 조합은 예상 상호 작용 파트너의 확인 (그림 2)을 용이하게한다. 또한, MS의 분석과 함께 알 수없는 상호 작용 파트너의 식별 (그림 3)?...

단백질의 기능을 이해하기 위해 그것의 파트너 상호 작용을 아​​는 것이 필수적이다. 여러 고전적인 기술은 수용성 단백질의 상호 작용 파트너의 ID를 사용할 수 있습니다. 그러나, 이러한 기술은 소수성 4에 의한 막 단백질을 쉽게 양도 할 수 없습니다. 이 한계를 극복하기 위해, 우리는 막 연쇄상 구균 단백질 상호 작용 실험 (막 척추) 6을 개발했습니다. 그것은 수용성 단백질 7 만 적합한 척추 방법을 기반으로합니다. 가교제의 포름 알데히드의 두 가지 장점에서 막 척추의 장점은 첫째, 포름 알데히드는 쉽게 세포막을 통과하기 때문에 살아있는 세포 8 interactome의 정확한 스냅 샷을 생성 할 수 있습니다. 둘째, 포름 알데히드 상호 링크는 9 끓는으로 되돌릴 수 있습니다. 여기서, 이러한 두 가지 이점은 멤브레인 PROTE 영구 아니라 과도 프로톤 펌프 억제제뿐만를 식별하는 데 사용기능 6. 요컨데, 패 혈성-태그가 일체형 멤브레인 미끼 단백질의 C-말단에 융합된다. 막 미끼 단백질을 발현하는 세포는 간 링크가 막 미끼 단백질로 단백질을 먹이로 포름 알데히드 (그림 1)와 함께 배양된다. 먹이 단백질의 수정은 필요하지 않습니다. 다음에, 막 분획을 제조하다. 따라서, 막 단백질은 단백질이 친화력 정화를 먹이 단백질과 공동으로 정제되어 세제 처리 및 미끼에 의해 용해된다. 이어서, 가교가 비등하여 반전되고, 미끼 및 그 공동 용출 먹이 단백질은 SDS-PAGE에 의해 분리된다. 마지막으로, 먹이 단백질은 면역 블롯 분석 또는 질량 분석법에 의해 확인 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="878">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">37% Formaldehyde</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">4979.1</td>
			<td style="width:271px;">Should not be older than one year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DNaseI</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:137px;">DN25</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BCA protein assay kit</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">23225</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Micromagnetic rod</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">0955.2</td>
			<td style="width:271px;">5 mm in length, 2 mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Triton X-100</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">6683.1</td>
			<td style="width:271px;">standard detergent for solubilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">n-Dodecyl-&#946;-maltoside</td>
			<td style="width:128px;">Glycon</td>
			<td style="width:137px;">D97002-C</td>
			<td style="width:271px;">the best detergent to solubilze CpxA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">1 ml Strep-Tactin superflow gravity flow column</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1207-050</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Strep-tag protein purification buffer set</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1002-001</td>
			<td style="width:271px;">Contains buffer W and buffer E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Amicon Ultra-4 centrifugal filter</td>
			<td style="width:128px;">Millipore</td>
			<td style="width:137px;">UFC803024</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">SuperSignal West Pico Chemiluminescent substrate</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">34080</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SilverQuest Silverstaining kit</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:137px;">LC6070</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FireSilver Staining Kit</td>
			<td style="width:128px;">Proteome Factory</td>
			<td style="width:137px;">P-S-2001</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ultracentrifuge</td>
			<td style="width:128px;"></td>
			<td style="width:137px;"></td>
			<td style="width:271px;"></td>
		</tr>
	</tbody>
</table>

membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo

막 단백질은 세포 생존에 필수적이며, 따라서 중요한 치료 표적 1-3이다. 그들은 단지 4에서 작동하기 때문에, 상호 작용을 파악하고 특징 짓는 방법은 5 필요합니다. 이를 위해, 우리는 막 척추 6라고 불리는 막 연쇄상 구균 단백질 상호 작용의 실험을 개발했다. 이 기술은 연쇄상 태깅 막 미끼 단백질의 친화도 정제하여 리버시블 가교제 포름 알데히드를 사용하여 가교 생체 내에서 결합한다. 절차 중에 가교 먹이 단백질은 막 단백질을 미끼로 공동 정제이어서 비등에 의해 분리된다. 막 등뼈를 사용하여 막 단백질의 단백질 - 단백질 상호 작용 (프로톤 펌프 억제제)를 분석 할 때 따라서, 두 가지 주요 작업을 실행할 수 있습니다 : 첫 번째, 면역 블, 그리고에 의해 제안 된 상호 작용 파트너의 확인 번째, 질량 분석 분석에 의해 새로운 상호 작용 파트너의 식별 . 또한,도 L친화력 아야, 과도 프로톤 펌프 억제제는이 기술에 의해 감지됩니다. 마지막으로, 막 척추는 막 단백질의 프로톤 펌프 억제제를 식별 할 수있는 강력한 검사 도구로 적용하고, 거의 모든 세포 유형에 적응할 수있다.

멤브레인 척추 분석은 막 단백질의 공동 정화 및 일시적으로 상호 작용 단백질 파트너를 허용한다. 공동 정제는 가교제 포름 알데히드를 사용함으로써 달성된다. 포름 알데히드 농도 및 가교 시간 : 두 매개 변수는 비특이적 교차 결합을 방지하기 위해 중요하다. 포름 알데히드의 과도한 충분하지만, 사용하지 않는 간단 면역 블 롯팅에 의해 제어 될 수있다. 포름 알데히드 가교 결합 된 단백질 복...

Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

생화학 적 방법은 막 단백질의 생체 내 단백질 - 단백질 상호 작용 (PPI)를 식별하기 위해 설명된다. 이 방법은 단백질 교차 결합, 친화력 정화 및 질량 분석을 결합하여 거의 모든 세포 유형이나 유기체에 적응할 수있다. 이 방법을 과도 프로톤 펌프 억제제의도 식별이 가능하게된다.

막 척추 : 막 단백질의 단백질 - 단백질 상호 작용을 식별 할 수있는 생화학 적 도구<em&gt; 생체</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

membrane-spine-biochemical-tool-to-identify-protein-protein

Research

JoVE Journal

Bioengineering

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

13.6K 조회수.  Universität Osnabrück.  생화학 적 방법은 막 단백질의 생체 내 단백질 - 단백질 상호 작용 (PPI)를 식별하기 위해 설명된다. 이 방법은 단백질 교차 결합, 친화력 정화 및 질량 분석을 결합하여 거의 모든 세포 유형이나 유기체에 적응할 수있다. 이 방법을 과도 프로톤 펌프 억제제의도 식별이 가능하게된다. 

비디오: Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

핵산, 단백질과 작은 분자의 Reagentless 감지를위한 전기 -의 DNA Biosensors의 제작

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

연구

생체공학

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

부정적 얼룩 EM에 의해 단백질과 Macromolecular의 단지를 떠올리 : 그리드 준비에서 이미지 수집

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

태반의 맞은 편에 생체이 물과 나노 물질의 전송 속도의 결정을 사용하여<em&gt; 생체</em&gt; 인간 태반 관류 모델

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

단백질 A 멤브레인 흡착제 및 간소화된 워크플로우를 사용하여 키메라 인간 Fc 발현 단백질의 격리 및 특성화

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

발광 공명 에너지는 포유 동물 세포에 표현 막 단백질의 구조 변화를 연구에 전송

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

위한 도구로 리포좀 캡슐화 된 근적외선 형광의 형광 담금질<em&gt; 생체</em&gt; 광학 이미징

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

샌드위치 같은 미세 환경은 셀 / 재료의 상호 작용을 활용하는

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Semiflexible 고분자 공부를 다양 한 도구로 DNA 나노튜브

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...