본 연구는 재향 군인의 미국학과에서 우수상 지원하고, NIH는 RW하는 R01CA138528을 부여했다.

식 구조의 1 준비 <ol><li> 프로토콜을 시작하기 전에, N-말단 에피토프 태그 (Myc와 또는 HA), 및 박테리아 세포에서 높은 수준의 단백질 발현 및 정제를위한 기판에서 프레임 글루타치온-S-전이 효소 (GST)와 함께 포유 동물 발현 벡터에 클론 PRMTs 표준 분자 생물학적 기술을 이용하여 용 해물. </li></ol> PRMTs의 2 정제 <ol><li> 인간 기관지 상피 세포 (Beas2B)을 유지하기 위해 100mm 배양 접시를 사용합니다. 통로 세포를 3 일마다 그들을 합류로 성장할 수 있도록하지 않습니다. </li><li> 형질 감염 전날에, 100mm 배양 접시에 10 ml의 배지에 7.5 × 105 세포를 시드. 소용돌이 균일하게 세포를 분산하고, 5 % 가습 CO 2 배양기에서 37 ° C에서 배양한다. </li><li> 다음날, 미디어를 대기음 혈청과 항생제없이 배양 배지 5 ㎖를 추가합니다. 소용돌이와 매체를 대기음. 이 단계를 반복 한더 많은 시간, 최종적 혈청 / 항생 배지 5 ㎖를 추가로 진행 형질. </li><li> 멸균 된 1.5 ㎖의 플라스틱 원심 분리기 튜브에서 무 혈청 배지 750 μL로 플라스미드 DNA 6 μg을 희석. </li><li> 또 다른 1.5ml의 플라스틱 원심 분리기 튜브에서, 무 혈청 배지 750 μL로 형질 감염 시약 30 μL 희석. </li><li> 희석 DNA (단계 2.4)을 함유하는 튜브로 희석 형질 감염 시약 용액 (단계 2.5)을 전송. 피펫 팅에 의해 여러 번 아래로 부드럽게 섞는다. </li><li> 15 분 동안 실온에서 형질 전환 혼합물을 배양한다. </li><li> 15 분 후, 세포 위에 가볍게 형질 전환 혼합물을 피펫, 소용돌이 균일하게 혼합하고, 5 % 가습 CO 2 배양기에서 37 ° C에서 배양한다. </li><li> 3 시간 후, 세포로부터 형질 전환 혼합물을 함유하는 배지를 흡인. 혈청과 항생제로 보충 신선한 세포 배양 배지를 추가합니다. </li><li> 5 % 가습 CO 2에서 37 ° C에서 품어48 시간 동안 인큐베이터. </li><li> 48 시간 후, 용해 완충액 1 ㎖로 세포를 용해 (1X 인산 완충 생리 식염수, 1 % Nonidet P-40, 0.5 % 나트륨 데 옥시 콜레이트, 0.1 % 나트륨 도데 실 설페이트, 1 μg / ml의 phenlymethyl 술 포닐 플루오 라이드). 셀 리프터와 플라스틱 원심 분리 튜브에 수집하고, 용 해물을 30 분 동안 얼음 위에서 배양 용 해물. </li><li> 세포 파편에서 해물을 취소 벤치 탑 원심 분리기에서 10 분 동안 15,000 XG에 해물을 원심 분리기. [* 이것은 고도로 메틸화 분석 용 효소의 정제를 진행하기 전에 항-HA 항체와 해물의 분취 량 면역 블로 통해 HA-PRMT1의 발현을 확인하기 위해 추천된다.] </li><li> PRMTs의 정화를 들어, 인산염 완충 생리 식염수에 항-HA 친 화성 기질의 50:50 현탁액 30 μL (PBS)와 세포 용 해물의 3 mg의 배양 및 튜브 회전에서 4 ° C에서 하룻밤 회전합니다. </li><li> 다음 날, 수집 2 분 동안 벤치 탑 원심 분리기에서 14,000 XG에서 튜브를 회전구슬. 뜨는을 취소하고이 14,000 XG에서 원심 분리 씻어 비즈 [20 mM 트리스 (산도 8.0), 150 mM의 염화나트륨, 5 mM의 EDTA, 1 % 트리톤 X-100] 각 버퍼 RIPA의 1 ㎖로 3 배 씻어 분. 셋째 세척 한 후, 메틸화 버퍼 100 ㎕ 한 번 구슬을 씻는다 (50 mM 트리스의 pH 8.5, 20 mM의의 KCl, 10 mM의 MgCl2를, 1 ㎜의 β-머 캅토 에탄올, 100 MM의 자당). 원심 분리 후, 상층 액을 제거합니다. 고정화 PRMTs 함유 구슬 메틸화 분석에 사용될 준비가되었다. [*이 높은 메틸화 분석에 즉시 고정 PRMTs를 사용하는 것이 좋습니다.] </li></ol> 기판의 3 정화 <ol><li> GST-G3BP1의 단일 콜로니가 100 μg / ml의 암피실린을 함유하는 루리아 - 베르 타니 (LB) 배지 5 ㎖에 접종 BL21 변형. 250 rpm에서 진탕 37 ° C에서 하룻밤 문화를 성장. </li><li> 다음 날, 신선한 LB 배지 및 계속 50 ㎖에 하룻밤 문화를 전송inue는 (그것은 약 2 시간 소요) OD 0.5-0.7의 600로 증가합니다. </li><li> OD 600이 0.5 ~ 0.7에 도달 할 때, 250 rpm에서 진탕 37 ° C에서 추가로 4 시간 동안 배양 한 mm의 최종 농도 이소 프로필-β-D-티오 갈 락토 시드 (IPTG)를 추가하고 계속하십시오. </li><li> 10 분 동안 1,500 XG에서 원심 분리하여 세포를 수확하고, -80 ° C에서 GST 정화 또는 저장소의 펠렛을 처리합니다. </li><li> 용해 완충액 5 ㎖의 세균 펠릿 다시 중단 (단백질 분해 효소 억제제, 라이소자임의 100 μg / ml의와 1X PBS를 0.1 % 트리톤 X-100) 및 30 분 동안 37 ° C에서 해물을 품어. 이 때 해물 인해 세균 게놈 DNA의 출시로 혼탁 한 점성이 나타납니다. </li><li> 초음파에 의한 게놈 DNA (1 초 30 %의 진폭을 가진 각각의 10 펄스) 전단. </li><li> 10 분 동안 15,000 XG에서 원심 분리 용 해물을 취소하고 15 ML 튜브에 맑은 상층 액을 전송합니다. 상층 액이 명확하지 않은 경우 centrifu를 반복gation 과정을 한 번 더. </li><li> 한편, 얼음 차가운 PBS로 두 번 GST-세 파로스 비드를 세척하고 PBS에서 GST-세 파로스 50:50 서스펜션을합니다. 삭제 박테리아 세포 용 해물에 GST-세 파로스 현탁액 200 μl를 추가하고 1 시간 동안 4 ° C에서 회전합니다. </li><li> 1 시간 후, 비드를 수집하는 3 분 동안 500 XG에서 튜브를 원심 분리. 구슬이 얼음 차가운 PBS로 3 배 씻으십시오. </li><li> 최종 세척 한 후, 용출 버퍼 (50 mM 트리스 산도 8.0, 150 밀리미터의 NaCl, 30 mM의 글루타티온) 100 ㎕에 구슬을 재현 탁하고 10 분 동안 4 ° C에서 회전합니다. </li><li> 2 분 동안 15,000 XG에 튜브를 원심 분리기 및 다른 플라스틱 원심 분리 관에 정제 된 단백질을 포함하는 상층 액을 전송합니다. 정제 된 단백질의 농도를 추정하기 위해 소 혈청 알부민 (BSA)의 공지 된 양 함께 정제 된 단백질의 5 μL를 SDS 폴리 아크릴 아미드 겔 전기 영동 (SDS-PAGE) 분석을 수행한다. </li></ol> 4 메틸화 분석 주 : 방사성 동위 원소 작업 및 방사성 동위 원소 실험을 수행하기에 기관의 프로토콜을 준수 지정된 별도의 영역에서 다음 단계를 수행합니다. <ol><li> 정제 된 GST-기판과 1X 메틸화 버퍼 (50 mM 트리스의 pH 8.5, 20 mM의의 KCl, 10 mM의 MgCl2를, 1 ㎜의 β-머 캅토 에탄올, 100 MM의 자당)의 1 μCi 포함하는 마스터 믹스의 2 μg 추가 S를 - adenosyl- L - [메틸 3 H] 메티오닌, 물 고정 PRMTs (단계 2.13)에 10 μL의 최종 볼륨과 1 시간 동안 30 ° C에서 반응을 품어. </li><li> 1 시간 후, 2X SDS 샘플 완충액 10 μl를 첨가하여 반응을 정지. 10 분 동안 95 ° C에서의 시료의 변성 후, SDS-PAGE 겔에서 샘플을 구분합니다. </li><li> 나중에, 반 건조 전기 전송을 사용하여 니트로 셀룰로오스 막에 SDS 겔에서 분리 된 단백질을 전송합니다. </li><li> 전사 후, 함께 막을 덮utofluor (방사 법적 화상 압기)가 부드럽게 실온에서 2 시간 동안 흔들어. 2 시간 후, autofluor을 부어 나중에 사용하기 위해 보관합니다. [* 방사성 물질로 튜브를 레이블] </li><li> 빨리 공기는, 여과지를 건조 막 플라스틱 백에 밀봉하고 -20 ° C에서 X-선 필름에 노출을 막. 충분한 노출 시간 5~30일 사이의 범위. </li></ol> PRMT 식 및 기판의 평등로드의 5 확인 <ol><li> 충분한 노출을 달성 한 후, 차단 버퍼가 포함 된 플라스틱 상자 [트윈 20 (TBST + 3 % 무 지방 우유)과 트리스 완충 식염수를]에 멤브레인을 전송하고 부드럽게 한 시간 동안 바위. </li><li> 방사성 폐기물로 차단 버퍼를 폐기하십시오. 사에서 밤새 TBST에서 : (1000 희석 1) 항-HA 항체와 함께 막을 배양 부드러운 락과 ° C,. </li><li> TBST 배와 멤브레인을 세척하고 HRP 표지 항 마우스 이차 항체로 막을 배양부드러운 락을 1 시간 동안 실온에서 TBST에서 (한 5,000 희석). </li><li> 1 시간 후, TBST 배와 멤브레인을 세척하고, HRP의 기질과 단백질의 chemiluminiscence 검출을 수행합니다. </li><li> HA-태그 PRMTs의 검출 후, 실온에서 5 분 동안 Ponseau-S 염색 용액과 함께 막을 배양한다. 신속하게 GST-태그 기판을 감지하는 물을 막 배를 씻는다. 이미지를 스캔합니다. </li></ol>

<ol>
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저자가 공개하는 게 없다.

본원에 기재된 프로토콜은 통상적으로 식별 PRMT 기판의 메틸화 상태를 설정하는 데 사용된다. 이 강력하고 일관성있는 분석은 또한 기판에 대한 PRMTs의 특이성에 대한 결정적인 증거를 제공 할 것입니다. 이 분석의 성공을위한 핵심 구성 요소는 다음과 같습니다 포유 동물 세포에서 PRMTs의 1 식, 정제 PRMTs의 2 활동, 3 식 및 기판의 정제 및 니트로 셀룰로오스 막에 단백질의 4 완료 서부 전송. <p cl...

단백질의 메틸화는 첫째 1968 일에 설명했다. 그것은 연구자들이이이 번역 후 변형의 중요성을 인식하기 시작, 1996 년 PRMT1의 첫 번째 복제까지되지 않았습니다. 흥미롭게도, 핵 추출물의 단백질 아르기닌 잔기의 약 2 %는이 수정의 풍요 로움을 나타내는 3 메틸화된다. 아르기닌 될 수 아르기닌 측쇄 내의 염기성 측쇄와 질소 / s와 양전하를 띤 아미노산 포스트 병진 메틸기, 아르기닌 메틸화 4-6로 알려진 프로세스를 추가로 변경됨. 아르기닌 메틸화 효소 즉의 클래스에 의해 촉매된다. 단백질 아르기닌 메틸 트랜스퍼 (PRMTs). 아르기닌은 monomethylated 또는 dimethylated 후자는 공정 4-6 PRMTs 촉매의 종류에 따라 대칭 또는 비대칭이 될 수 될 수 있습니다. arginin 표시 단백질전자 글리신이 풍부한 모티브 PRMT 매개 촉매에 대한 잠재적 인 대상이다. 기판 PRMT 매개 메틸화 정상 세포 항상성 7-9에 중요한 모두의 단백질 - 단백질 상호 작용, 단백질 - 핵산 상호 작용, 단백질 기능, 유전자 발현 및 / 또는 세포 신호 전달을 조절하는 것으로 나타났다. 단백질 아르기닌 메틸화의 생물학적 역할을 이해하기 위해서는, 정확한 효율과 재현성이 분석법은 식별 PRMT 기판의 메틸화 상태를 설정하기 위해 필요하다. 자신의 기판의 메틸화를 촉진하기 위해 정제 PRMTs의 능력을 평가하는 시험 관내 메틸화 분석에서, 아르기닌 메틸화 10-12 연구를위한 잘 받아 분석이다. 이 분석의 전반적인 성공은 대부분 정제 된 PRMTs의 활동에 따라 달라집니다. PRMTs 표현 박테리아 나 포유류 세포 10,11로부터 정제 할 수있다. D 등이 체외 메틸화 분석,프로토콜 절에 etailed 원래 티니와 동료 (10)에 의해 설명 된 방법에 기초한다. 이 프로토콜에서는 상세히 PRMT1 포유류 세포에서의 발현 및 정제하는 단계를 나타낸다. 단백질 결합 단백질 1 (G3BP1), 공지 PRMT 기판 (8)를 활성화하는 라스-GTPase에 메틸화를 촉진하는 정제 PRMT1의 능력은 이상과 같은 S-아데노 실-L-[메틸 3 H] 메티오닌의 존재하에 평가 하였다 메틸 공여체. 이 분석을 사용하여, 우리는 확실하게 단백질 아르기닌 메틸화 연구에서 주요 단계 메틸 레이트 신규 또는 공지 된 기판에 PRMTs의 능력을 정의 할 수있다.

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			<td height="21" style="height:21px;">Bottle top dispenser</td>
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			<td height="21" style="height:21px;">6 ml Scintillation Vials</td>
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			<td height="21" style="height:21px;width:259px;">Sodium deoxycholate</td>
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			<td height="21" style="height:21px;width:259px;">PMSF</td>
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			<td style="width:111px;"></td>
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			<td height="21" style="height:21px;width:259px;">Nacl</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
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			<td height="21" style="height:21px;width:259px;">Bio-rad gel doc</td>
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			<td height="21" style="height:21px;width:259px;">Ponseau S</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
	</tbody>
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in vitro methylation assay to study protein arginine methylation

단백질 아르기닌 메틸화는 핵에서 가장 풍부하게 번역 후 변형 중 하나이다. 단백질 아르기닌 메틸화는 식별 및 / 또는 프로테오믹스 방법을 통해 결정, 및 / 또는 메틸 아르기닌 특이 항체 발현 수준으로 할 수있다. 그러나, 이러한 기술은 때때로 오해의 소지가 될 수 있으며 종종 거짓 긍정적 인 결과를 제공한다. 가장 중요한 것은, 이들 기술은 메틸화 분석법은, 반면에, 민감한 유용한 생화학 분석법이다 체외. PRMT의 기질 특이성을 지원하는 직접적인 증거를 제공 할 수 있고, 식별 된 단백질이 실제로 PRMT 기판 인 경우 일관 공개. 전형적인 관내 메틸화 분석은 정제 된 활성 PRMTs 정제 기판과 방사성 동위 원소 표지 된 메틸 공여체 (S-아데노 실-L-[메틸 3 H] 메티오닌)을 포함한다. 여기에서 우리는 촉매 활성 PRMT1, 재하 표현 PRMT의 가족을 분리하는 단계별 프로토콜을 설명합니다. 나정제 PRMT1의로 메틸 트랜스퍼 활동 이후 S-아데노 실-L-메틸 공여체로서 [메틸 3 H] 메티오닌의 존재하에, 단백질 1 (G3BP1)를 결합 라스-GTPase 활성화 단백질, 공지 PRMT 기판 상에 시험 하였다. 이 프로토콜은 새로운 생리 PRMT1 기판의 메틸화 상태를 설정하기위한,뿐만 아니라 단백질 아르기닌 메틸화의 기본 메커니즘을 이해하기 위해 단지 사용될 수있다.

<html> GST-G3BP1 메틸화하는 HA-PRMT1의 능력은 체외 메틸화 분석에 의해 결정되었다. Beas2B 세포 용 해물로부터 정제 HA-태그 PRMT1는 GST-G3BP1를 포함하는 메틸화 반응에 고용되었다 메틸 공여체 S-아데노 실-L-[메틸 3 H] 메티오닌 1 μCi. PRMT1 효율적 GST-G3BP1 (그림 1, 상단 패널)의 메틸화를 촉진 할 수있다. 음성 대조군 (그림 1, 상판, 차선 1)을 역임 빈 벡터 형질 Beas2B 세?...

Watch this Scientific Journal Video about In vitro Methylation Assay to Study Protein Arginine Methylation at JoVE.com

In vitro Methylation Assay to Study Protein Arginine Methylation

단백질 아르기닌 메틸화, 즉 효소의 클래스에 의해 촉매. 단백질 아르기닌 메틸 트랜스퍼 (PRMTs)는 단백질 내의 아르기닌에 메틸기 (들)의 첨가의 효소 과정이다. 체외 메틸화 분석은 공지 또는 신규 PRMT 기판의 메틸화 상태를 평가하기위한 가장 신뢰할 도구이다.

체외 메틸화 분석에서 단백질 아르기닌 메틸화를 공부합니다

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

in-vitro-methylation-assay-to-study-protein-arginine-methylation

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Biology

In vitro Methylation Assay to Study Protein Arginine Methylation

15.1K 조회수.  University of Illinois at Chicago.  단백질 아르기닌 메틸화, 즉 효소의 클래스에 의해 촉매. 단백질 아르기닌 메틸 트랜스퍼 (PRMTs)는 단백질 내의 아르기닌에 메틸기 (들)의 첨가의 효소 과정이다. 체외 메틸화 분석은 공지 또는 신규 PRMT 기판의 메틸화 상태를 평가하기위한 가장 신뢰할 도구이다. 

비디오: In vitro Methylation Assay to Study Protein Arginine Methylation

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

에 각인 유전자의 DNA Methylation의 결정 Arabidopsis 배젖

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

단백질 막 중첩 분석 : 가용성식이 및 불용성 단백질 간의 상호 작용을 테스트하는 프로토콜 체외에서</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

의 DNA Methylation : Bisulphite의 변경 및 분석

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

단백질 - 단백질 상호 작용 연구를 자성 비즈를 사용하여 액상 동질 캡처 분석 : Poliovirus-Nanobody의 예

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

의 적용<em&gt; 체외</emDPS 단백질의 스트레스 중재 속성을 시각화&gt; DNA 보호 분석

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

에 대한 내피 세포의 관 형성의 분석<em&gt; 체외</em&gt; 혈관 신생 연구

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

자료 쌍 해상도에서 DNA 메틸화의 평가에 대한 표현 바이 설 파이트 시퀀싱을 감소 향상

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.