우리는하고 싶은 AV로 Lefkofsky 가족 재단 / 리즈와 에릭 Lefkofsky 혁신 연구 상 - 여기에 설명이 프로젝트는 NIH / NCI (NCI-R01CA182506-01A1)에서 부여에 의해뿐만 아니라 로버트 H. 루리 종합 암 센터 지원 중요한 읽기와이 원고를 편집하기위한 실험실의 구성원 (캐롤 O&#39;Callaghan와 엘리자베스 앤 웨인) 감사합니다.

체외 탈 아세틸 분석 1. <ol><li> SIRT2의 정제 <ol><li> 10 % FBS와 항생제 8 ML의 DMEM에서 배양하고 70 % 합류 다음날로 37 ° C, 5 % CO 2 조직 배양 인큐베이터에서 배양하는 HEK 293T 세포를 10㎝ 배양 플레이트를 준비한다. </li><li> 다음 날, 공동 트랜 4 μg의 pCDH - 순수하지 않아-GFP-SIRT2-국기 (우리의 실험실에서 만든 lentivector), 4 μg의 pCMV- dR8.2 dvpr (포장 벡터) 0.5 μg의을 pCMV-VSV-G (봉투 벡터) 3 μL PEI / μg의 DNA의 비율 18 사용 폴리에틸렌 이민 (PEI). </li><li> 12 시간 후 미디어를 변경합니다. </li><li> 36-48 시간 후 상층 액을 수집합니다. </li><li> 4 ° C에서 5 분 동안 1,000 XG에서 원심 분리하여 이물질을 제거합니다. </li><li> 0.22 μm의 필터를 통해 여과 한 후, SIRT2의 렌티 바이러스 1 ml의 분취 량을 -80 ℃에서 보관하십시오. </li><li> 바이러스를 사용하지 않는 경우에 녹여 얼음 SIRT2 렌티은 (는 -80 ° C에서 바이러스를 저장하는 것이 중요). </li><li> 8 μg의 / ㎖의 폴리 브렌의 존재 렌티 1 ㎖로 80~90% 합류 된 HEK 293T 세포를 6 웰 플레이트를 감염시킨다. </li><li> 다음 날 (24 시간)까지 37 ° C 배양기에서 바이러스에 감염된 세포를 놓습니다. </li><li> 24 시간 후 배지를 교체합니다. </li><li> 감염 후 48 시간은 형광 현미경으로 GFP를 양성 세포를 감지하여 감염의 효율성을 확인합니다. </li><li> 감염 효율이 좋은 경우에 (적어도 40-50% 감염된 세포)은 배양 배지를 세포를 발현하는 안정적인 SIRT2 대한 선택이 μg의 / ml의 퓨로 마이신을 함유하는 배지를 교체한다. 주 :이 약 2 주가 소요 및 배지를 3-4 일마다 변경된다. 비 감염된 세포는 SIRT2의 렌티 바이러스 성장을이 선택 기간에만 감염된 세포를 통해 죽는다. </li><li> 을 정제를 시작하기 전에 10 % SDS-PAGE 겔상에서 총 단백질 40 μg의 실행 후 (1000 희석 1) 항 - 플래그 항체를 사용하여 웨스턴 블롯에 의해 플래그 - 태그 SIRT2 발현 분석ication 과정 (권장) 19, 20. </li><li> 1 활성 SIRT2, 분할 세포를 정화하려면 안정적으로 국기 - SIRT2를 과발현 HEK 293T 세포의 충분한 요리를하기 위해 트립신 3 비율 (텐 10cm 요리 후속 실험에 대한 충분한 단백질의 정제를 수 있습니다). <ol><li> 상기 세포를 Trypsinize 배지를 제거하고 멸균 DPBS로 세포를 세척하여 잔류 혈청을 제거하기 위해. 천천히, 세포 단층을 포함 30 ~ 45 초 동안 상온에서 부화 0.25 % 트립신 - EDTA 용액 2.5 ml를 추가합니다. 세포가 분리 시작할 때까지 트립신 - EDTA 용액을 제거한 후, 37 ° C에서 품어. 그런 문화 매체를 추가하고 새로운 요리에 세포를 전송합니다. </li></ol></li><li> 4 ° C에서 10 분 동안 1,000 XG에서 원심 분리 트립신 처리하여 세포를 PBS로 두 번 세포를 씻어 수집, 배지를 제거합니다. </li><li> 500 ㎕의 용해 완충액 (20 mM의 HEPES pH가 7.9, 18 mM의 KCl을 0.2 mM의 EGTA, 1.5 밀리미터의 MgCl 2를 Lyse 세포 펠렛, 프로테아제 억제제 칵테일과 일정한 회전에서 4 ° C에서 30 분 동안 1 μM TSA를 포함하여 20 % 글리세롤, 0.1 % NP-40). </li><li> 새로운 튜브 15 4 ° C에서 최소 및 전송 상층 액 14,000 XG에 원심 분리기. </li><li> 후술하는 바와 같이 아가 로스 비드를 결합하는 항 - 플래그 항체를 사용하여 면역 침전 법을 수행한다. <ol><li> 단백질 분해물 (10 ~ 20 mg의 총 단백질)에 구슬 아가로 오스 복합 안티 - 플래그 항체의 200 ~ 300 μl를 추가합니다. </li><li> 일정한 교반과 함께 4 ° C에서 밤새 품어. </li><li> 4 ℃에서 5 분 동안 1,000 XG에서 원심 분리하여 immunoprecipitates를 수집합니다. </li><li> 4 ℃에서 5 분 동안 1,000 XG에서 펠렛을 10 ml의 용해 버퍼 5 회 반복한다. 각 세척 한 후, 비드 펠렛 및 버퍼 A.으로 뜨는 교체 </li></ol></li><li> (프로테아제 억제제 칵테일 1 PBS에서 μm의 TSA 포함) 1 배 플래그 펩타이드 솔루션을 준비합니다. </li><li> 1 배 플래그 peptid 500 μl를 추가아가로 오스 비즈에 전자 솔루션 및 일정 회전에서 4 ° C에서 30 분 동안 품어. </li><li> 4 ℃에서 2 분 동안 6,000 XG에서 원심 분리하여 상층 액을 수집합니다. </li><li> 앞의 두 단계를 반복합니다. </li><li> 4 ° C에서 1 분 동안 15,000 XG에 필터 튜브와 원심 분리를 이용하여 상층 액에서 구슬을 제거합니다. </li><li> 최종 단백질 농도를 1 μg의 때까지 / 한외 여과막을 사용하여 용출 μL 샘플을 농축시킨다. </li><li> 용출 된 시료의 1 μg의를 사용하여 수행하는 전기 영동으로 정제 과정을 확인합니다. </li><li> 시중에서 판매하는 염색 액에 얼룩 젤 SIRT2 정화를 확인합니다. </li><li> -80 ° C에서 정제 SIRT2을 유지합니다. </li></ol></li><li> 아세틸 단백질 기판의 정제 <ol><li> 약 70 %의 합류 다음날로 HEK의 293T 세포를 10 ㎝의 배양 접시를 준비합니다. </li><li> 국기의 5 μg의와 다음 날, 공동 트랜 세포는 플라스미드 벡터 expressin 태그g 단백질 기질뿐만 아니라 3 μL PEI / μg의 DNA의 비율로 PEI를 사용하여 단백질의 아세틸 화 수 특정 아세틸 트랜스퍼 5 μg의. 주 : 특정 아세틸 트랜스퍼 라제는 공지되어 있지 않은 경우, 예컨대 P300, CBP, GCN5, Tip60과 PCAF 공지 히스톤 아세틸 트랜스퍼 (HAT들)의 혼합물로 공동 트랜. </li><li> 12 시간 후 매체를 변경합니다. </li><li> 일 세포를 용해시키기 전에, 아세틸 라제를 억제하여 단백질 - 기재의 아세틸 화 수준을 극대화하는 TSA / NAM을 포함하는 배지로 배양액을 대체하여 밤새 1 μM의 TSA 2 mM의 니코 티나 마이드 (NAM)로 세포를 처리한다. </li><li> 48 시간 형질 전환 한 후, 배지를 제거 4 ℃에서 10 분 동안 1,000 XG에서 원심 분리 트립신으로 세포를 PBS로 두 번 세포를 씻어 수집합니다. </li><li> 접시 당 500 ㎕의 용해 완충액에 세포를 Lyse 펠릿 (20 mM의 HEPES pH가 7.9, 18 mM의 KCl을 0.2 mM의 EGTA, 1.5 mM의 MgCl2를, 20 % 글리세롤, 0.1 % NP-40) 프로테아제 I을 포함nhibitors 칵테일, 1 μM의 TSA와 일정한 회전에서 4 ° C에서 30 분 동안 2 MM의 NAM. </li><li> 새로운 튜브 15 4 ° C에서 최소 및 전송 상층 액 14,000 XG에 원심 분리기. </li><li> 후술하는 바와 같이 아가 로스 비드를 결합하는 항 - 플래그 항체를 사용하여 면역 침전 법을 수행한다. <ol><li> 단백질 분해물 (10 ~ 20 mg의 총 단백질)에 구슬 아가로 오스 복합 안티 - 플래그 항체의 200 ~ 300 μl를 추가합니다. </li><li> 일정한 교반과 함께 4 ° C에서 밤새 품어. </li><li> 4 ℃에서 5 분 동안 1,000 XG에서 원심 분리하여 immunoprecipitates를 수집합니다. </li><li> 4 ℃에서 5 분 동안 1,000 XG에서 펠렛을 10 ml의 용해 버퍼 2 회 반복한다. 각 세척 한 후, 비드 펠렛 및 용해 버퍼 A.으로 뜨는 교체 </li><li> 4 ° C에서 5 분 동안 1,000 XG에서 NAM없이 펠렛을 10 ml의 용해 버퍼 3 회 반복한다. 각 세척 한 후, 비드 펠렛 및 그것은 importa이다 용해 버퍼 A.으로 뜨는 교체NT를 탈 아세틸 화 반응에 대한 NAM를 이월 할 수 없습니다. </li></ol></li><li> (단백질 분해 효소 억제제와 PBS에서 1 μM TSA 포함) 1 배 플래그 펩타이드 솔루션을 준비합니다. </li><li> 아가로 오스 비즈로 1 배 플래그 펩티드 용액 500 μl를 추가하고 일정한 회전에서 4 ° C에서 30 분 동안 품어. </li><li> 4 ℃에서 2 분 동안 6,000 XG에서 원심 분리하여 상층 액을 수집합니다. </li><li> 앞의 두 단계를 반복합니다. </li><li> 4 ° C에서 1 분 동안 15,000 XG에 필터 튜브와 원심 분리를 이용하여 상층 액에서 구슬을 제거합니다. </li><li> 단백질 농도를 1 μg의 / UL 때까지 한외 여과막을 사용하여 용출 샘플을 농축시킨다. </li><li> 4-12 % SDS-PAGE 겔상에서 용출 시료 1 μL를 실행하여 전기 영동을 수행한다. </li><li> (1,000 희석 1) 단백질 기판 (희석 사용되는 특정 항체에 따라 다름) 서부 AC-K에 대한 항체를 사용하여 블로 팅에 의해 단백질 기판의 아세틸 화를 확인합니다. </li><li> 푸리 유지들이었다는 -80 ° C에서 단백질 기판을 아세틸. </li></ol></li><li> 체외 탈 아세틸 반응 <ol><li> 탈 아세틸 버퍼 B를 준비 (에 50 mM Tris-HCl, pH 7.5, 150 mM의 염화나트륨, 1 ㎜의 MgCl 2 0.5 μM TSA) </li><li> 20 ㎕의 최종 부피 (표 1)에 서로 다른 튜브에 3 개의 다른 반응을 준비합니다. </li><li> (선택 사항) SIRT2에 의해 단백질 기판의 탈 아세틸를 확인하기 위해 추가 반응을 포함합니다. (NAM 대신 아세틸 라제 활성 SIRT2의 반응 (pCDH - 순수하지 않아-GFP-SIRT2 H187Y -flag의 lentivector 사용 후이 1.1에 기술 된 프로토콜을 사용하여 수행 할 수 있습니다) 정제 탈 아세틸 널 (null) 돌연변이 SIRT2의 2 μg의 추가 또는 10 밀리미터에게 니코틴을 추가 ) SIRT2의 탈 아세틸 화 활성을 억제하는 반응 (표 1). </li><li> 일정하게 교반하면서 30 ℃에서 3 시간 동안 반응을 품어. </li><li> F 배 샘플 완충액 20 μl를 첨가하여 반응을 정지하고, 샘플을 비등또는 95 ℃에서 10 분. </li><li> 4-12 % SDS-PAGE 겔상에서 반응 혼합물을 실행하여 전기 영동을 수행하고 항 AC-K 항체 (1 : 1000 희석)를 사용하여 웨스턴 블롯에 의해 단백질 기판의 아세틸 화 상태를 검출 19,20한다. </li></ol></li></ol> 생체 탈 아세틸 분석 2. <ol><li> 배양 된 세포에서 과발현 SIRT2 <ol><li> HEK의 293T 세포와 10cm 배양 접시 안정적으로 pCDH - 순수하지 않아-GFP-빈 벡터를 과발현을 준비 pCDH - 순수하지 않아-GFP-SIRT2 - 플래그 및 pCDH - 순수하지 않아-GFP-SIRT2 H187Y -flag는 (1.1에서 설명) 70 일합니다 %의 합류. </li><li> 대안 적으로, 세포를 약 70 % 합류로 10cm 배양 접시를 준비하고, 3 μL PEI / μg의 DNA의 비율로 PEI를 사용하여 상기 언급 한 벡터 5㎍을 사용하여 일시적 과발현을 수행한다. </li><li> 국기-t의 과발현을 입증 (1,000 희석 1) 안티 - 플래그 항체를 이용하여 총 세포 추출물의 서쪽 블로 팅을 수행SIRT2 19 agged. </li></ol></li><li> 배양 된 세포의 최저 SIRT2에 RNA 간섭 <ol><li> 약 70 % 합류 다음날으로 HEK 293T 세포에의 10㎝ 배양 플레이트를 준비한다. </li><li> 다음 날, 공동 트랜 4 μg의 pLKO1 - 순수하지 않아 - 쉬 SIRT2 (lentivector) 19, 4 μg의 pCMV- dR8.2 dvpr (포장 벡터) 0.5 μg의을 pCMV-VSV-G (봉투 벡터) 18 사용 폴리에틸렌 이민 (PEI) 3 μL PEI / μg의 DNA가 비율. 참고 : SIRT2을 쓰러 뜨린를 들어, 우리가 RNAi의 컨소시엄 (TRC)가 설계 한 pLKO.1의 렌티 바이러스 벡터에 간단한 머리핀 shRNA를 사용합니다. </li><li> shSIRT2의 렌티 바이러스를 생산하고 SIRT2를 넉다운하는 표적 세포를 감염 1.1에 설명 된대로 절차를 따르십시오. </li><li> 10 % SDS-PAGE 겔에 총 단백질의 40 μg의를 실행 한 후 효율적인 SIRT2 최저를 보여 (1,000 희석 1) 항 SIRT2 항체를 이용하여 총 세포 추출물의 웨스턴 블로 팅을 수행합니다. </li><li> 또한,세포를 약 70 %의 합류로 10cm 문화 요리를 준비하고 제조업체의 지침에 따라 siRNA를를 사용 SIRT2의 과도 최저를 수행합니다. </li><li> 10 % SDS-PAGE 겔에 총 단백질의 40 μg의를 실행 한 후 효율적인 SIRT2 최저를 보여 (1,000 희석 1) 항 SIRT2 항체를 이용하여 총 세포 추출물의 웨스턴 블로 팅을 수행합니다. </li></ol></li><li> 면역 및 면역 블 롯팅은 배양 세포에서 아세틸 레벨을 감지하는 <ol><li> 12 시간 SIRT2를 과발현하거나 세포에서 세포 펠렛을 수집하기 전에 비 클래스 III 히스톤 아세틸 라제를 억제하는 배양 배지에 1 ㎛ TSA를 추가, (위의 2.1 참조) SIRT2 세포 (위 2.2 참조)를 무너 뜨렸다. </li><li> 4 ° C에서 10 분 동안 1,000 XG에서 원심 분리 트립신 처리하여 세포를 PBS로 두 번 세포를 씻어 수집, 배지를 제거합니다. </li><li> 10 ml의 용해 완충액 (20 mM의 HEPES pH가 7.9, 18 mM의 KCl을 0.2 mM의 EGTA, 1.5 추가밀리미터의 MgCl 2, 20 % 글리세롤, 프로테아제 억제제 칵테일, 1 μM의 TSA 2 MM의 NAM을 포함하여 0.1 % NP-40). </li><li> 일정한 회전에서 30 분 동안 4 ° C에서 품어. </li><li> 4 ℃에서 15 분 동안 20,000 XG에서 원심 분리하여 상층 액을 수집합니다. </li><li> 브래드 포드 (Bradford) 단백질 분석을 사용하여 단백질 농도를 계산한다. </li><li> 참고로 용해 완충액 A를 사용하여 표 2를 사용하여 1 ㎖의 최종 부피에서 새로운 전송 튜브 전체 단백질 1 mg의 하나 또는 과발현 SIRT2 SIRT2 최저 후의 세포로부터 단백질 샘플을 제조 하였다. </li><li> 단백질 A / G와 함께 특정 단백질 기판에 대한 항체를 사용하여 면역 침전을 수행합니다 (40 μl의 비드 슬러리 권장). 또한, 모든 아세틸 화 단백질을 면역 침전에 구슬을 아가로 오스 복합 항 AC-K 항체 (20 μl의 비드 슬러리는 일반적으로 충분하다)를 사용합니다. </li><li> 정전류 회로에서 4 ° C에서 샘플을 품어NT를 회전 하룻밤. </li><li> 4 ℃에서 2 분 동안 1,000 XG에서 원심 분리하여 immunoprecipitates를 수집합니다. </li><li> 4 ° C에서 2 분 동안 1,000 XG에 구슬 1 ml의 버퍼와 5 회 반복한다. </li><li> 배 샘플 버퍼의 40 μL에 재현 탁 구슬. </li><li> 95 ° C에서 10 분 동안 삶아 샘플. </li><li> 용출 샘플을 전송하는 동안 구슬을 방해하지 않고 4-12 % SDS-PAGE 겔에 용출 된 샘플 (단계 2.3.12에서 40 μl를) 실행하여 전기 영동을 수행합니다. </li><li> 단백질 기질에 특이적인 항체는 면역에 사용 된 경우 (1000 희석 1), 또는 단백질이 기판에 대해 특정 항체 (위한 권장 사항을 방지 AC-K 항체를 이용한 웨스턴 블롯으로 단백질 기판의 아세틸 화 수준을 감지 안티 AC-K 비드 면역에 사용 된 경우에 대하여 특이 적 항체 희석). </li></ol></li></ol>

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저자는 공개 아무것도 없어.

Recent high throughput proteomic studies have established acetylation as a widespread PTM found not only in nucleus but also in cytoplasm and mitochondria 7,8,21-23. Taking into account the likelihood that many more acetylated proteins and sites might have been not detected due to several reasons, such as specificity of the anti-Ac-K antibodies used, the low abundance of the acetylated proteins, and the transient nature of the PTM, it is safe to predict that more acetylated proteins remain to be discovered in ...

Post-translational modifications (PTMs) regulate cell signaling networks allowing cells to rapidly respond to internal and external signals. Over the last few decades, many different PTMs playing a pivotal role in diverse processes have been identified but only a few have been studied extensively, such as phosphorylation, acetylation and ubiquitination 1-3. Focusing on acetylation, Allfrey et al. were the first to propose a role for histone acetylation in regulating gene transcription about 50 years ago 4. Research in this field has revealed that histone lysine acetylation modulates chromatin condensation and it is considered to be an epigenetic mark as part of the histone code 5. Although it took a long time until the discovery of tubulin as the first non-histone acetylation target 6, it is well established now that hundreds of eukaryotic proteins beyond histones can be acetylated and lysine acetylation has been recognized as a wide-spread PTM that may rival phosphorylation and ubiquitination in its prevalence 7-9. Interestingly, non-histone acetylated proteins can be signaling molecules in the cytoplasm, transcription factors in the nucleus, and metabolic enzymes in mitochondria, highlighting the significance of acetylation in regulating a plethora of cellular processes. 
The acetylation status of a protein depends on the coordinated and opposing function of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) which add and remove acetyl groups from proteins. The reversible acetylation of lysine, which involves neutralization of a positive charge 10, alters protein structure and it seems very likely to also alter enzymatic function in several cases 11-13. Focusing on KDACs, 18 proteins have been identified in the human and mouse genomes 14-16. Among them, mammalian sirtuins (also called class III histone lysine deacetylases) which are distinct from other members as they require NAD+ for their enzymatic function, have attracted extensive interest in this research field 16. In mammals, seven sirtuins (SIRT1-7) have been identified, each of them sharing a conserved 275-amino-acid catalytic core domain, which are mainly categorized according to their subcellular localization to the nucleus (SIRT1, 6, and 7), mitochondria (SIRT3, 4, and 5), or cytoplasm (SIRT2). SIRT1-3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity, SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity but are involved in other types of acylations 17. In accordance with the significance of acetylation as a regulatory PTM modification involved in several cellular functions, sirtuins have also been implicated in a wide range of processes. After the first breakthrough studies establishing the role of sirtuins in life span extension, it has been shown that they are involved in diverse cellular functions including DNA repair, maintenance of genomic instability, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks and metabolism, as well as contributing to the initiation and/or progression of age-related diseases such as cancer, neurodegeneration and type 2 diabetes 15,16.
Despite the significant progress in the field of sirtuin biology, more work remains to unravel undiscovered roles and functions through the identification of novel substrates. This is evidenced more emphatically by recent advances in high-resolution mass spectrometry (MS) based proteomics which have significantly increased the number of proteins found to be acetylated but most importantly have identified several different acetylated lysines in each protein, arguing that acetylation may be as wide-spread as other PTMs such as phosphorylation 7,8,17. Taking into consideration that specific deacetylases have not yet been identified for most of these acetylated proteins-substrates, it is reasonable to suggest that both in vitro and in vivo deacetylation assays are needed to confirm and establish an acetylated protein as a legitimate substrate of a specific deacetylase. In the experimental protocols described below, details will be given on how to perform both in vitro and in vivo deacetylation assays using SIRT2 as the specific deacetylase.

<table border="0" cellpadding="0" cellspacing="0" width="1543">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">cell culture dishes</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">T1110 and T1115</td>
			<td style="width:728px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">pCDH-puro-GFP lentiviral vector</td>
			<td style="width:239px;">System Biosciences</td>
			<td style="width:119px;">CD513B-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV- dR8.2 dvpr (packaging vector)</td>
			<td style="width:239px;">Addgene</td>
			<td>8455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV-VSV-G (envelope vector)</td>
			<td style="width:239px;">Addgene</td>
			<td align="right">8454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylenimine (PEI)&#160;</td>
			<td>Polysciences Inc.</td>
			<td align="right" style="width:119px;">24885</td>
			<td>other transfection reagents can be used as well. PEI is cost effective and very efficient in transfecting 293T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22&#956;m filters</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">F5512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polybrene</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H9268</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">fluorescent microscope</td>
			<td style="width:239px;">Carl Zeiss MicroImaging Inc.</td>
			<td style="width:119px;">Axiovert 200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:239px;">Invivogen</td>
			<td style="width:119px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:239px;">Corning</td>
			<td style="width:119px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag antibody</td>
			<td style="width:239px;">Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">M9272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">EGTA</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">34596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protease inhibitors coctail 100x</td>
			<td style="width:239px;">Biotool</td>
			<td style="width:119px;">B14001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichostatin A (TSA)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T8552</td>
			<td>selective inhibitor of class I and II histone deacetylases (HDACs) but not class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag agarose beads</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:119px;">5417R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">rotator</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td style="width:119px;">415220Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">filter tubes</td>
			<td style="width:239px;">Millipore</td>
			<td>UFC30HV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flag peptide&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td>F3290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivaspin Centrifugal Concentrator</td>
			<td>Sartorius Stedim Biotech S.A.</td>
			<td>VS0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain solution&#160;</td>
			<td style="width:239px;">Invitrogen</td>
			<td style="width:119px;">LC6060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE LDS sample buffer (4x)</td>
			<td style="width:239px;">Life Techologies</td>
			<td style="width:119px;">NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Ac-K antibody</td>
			<td style="width:239px;">Cell Signaling</td>
			<td style="width:119px;">9441</td>
			<td>several anti-Ac-K antibodies are available. In our hands, the Cell Signaling antibody exhibits the highest sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl pH 7.5</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T5941</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NAD+</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">N0632</td>
			<td>required cofactor for sirtuins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nicotinamide (NAM)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">72340</td>
			<td>selective inhibitor class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pLKO.1 lentiviral vector&#160;</td>
			<td style="width:239px;">Addgene</td>
			<td style="width:119px;">8453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SIRT2 si RNA&#160;</td>
			<td style="width:239px;">Qiagen</td>
			<td style="width:119px;">GS22933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-SIRT2 antibody</td>
			<td style="width:239px;">Proteintech</td>
			<td style="width:119px;">15345-1-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bradford protein assay</td>
			<td style="width:239px;">BIO-RAD</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti Ac-K agarose beads</td>
			<td style="width:239px;">Immunechem</td>
			<td>ICP0388</td>
			<td></td>
		</tr>
	</tbody>
</table>

deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

단백질을 위해 시험 관내 및 생체 내 탈 아세틸 분석 모두에서 탈 아세틸 화 활성을 갖는 임의의 효소를 탈 아세틸 적법한 대상으로서 취급 할 아세틸 라제 및 기판 사이의 상호 작용을 확립하기 위해 수행 될 필요가있다. 분석이 완료되기 전에 시험 관내 아세틸 라제 분석을 위해, 아세틸 라제 및 아세틸 단백질 기질 모두의 정제가 필요하다. 여...

Watch this Scientific Journal Video about Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates at JoVE.com

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

탈 아세틸 화 분석법은 시르 투 인 사이의 상호 작용 (SIRT2) 및 특정 단백질 기판을 해명

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

deacetylation-assays-to-unravel-interplay-between-sirtuins-sirt2

Research

JoVE Journal

Chemistry

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

8.2K 조회수.  Feinberg School of Medicine, Northwestern University. This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

비디오: Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

종이 접기 고취 패턴 및 재구성 입자의 자기 조립

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

연구

화학

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

신속한 Colorimetric Assays는 질적 Biomolecular 샘플에서 RNA와 DNA를 구별 할

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

그람 음성 박테리아에서 분리 및 지질의 화학 특성

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

분할 및 풀 합성 펩타이드 급 아미드 도서관의 특성

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. Isonicotinic acid, or its derivatives, are coupled to variable length diaminoalkane chains under an inert atmosphere in anhydrous DMF or DMSO with the use of a weak base, triethylamine, and a coupling agent, 1-propylphosphonic anhydride. The products precipitate from solution upon formation or can be precipitated by the addition of water. If desired, the ligands can be further purified by recrystallization from hot water. Dinuclear platinum complex synthesis using the bispyridine ligands is done in hot water using transplatin. The most informative of the chemical characterization techniques to determine the structure and gross purity of both the bispyridine ligands and the final platinum complexes is 1H NMR with particular analysis of the aromatic region of the spectra (7-9 ppm). The platinum complexes have potential application as anticancer agents and the synthesis method can be modified to produce trinuclear and other multinuclear complexes with different hydrogen bonding functionality in the bridging ligand.

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

이핵 항암제로 플래티넘 비스 피리딘 계 리간드와 이들 착물의 합성 아미드 커플 링 반응

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

특성, 벤젠 폴리 카르 복실 산을 사용하여 발열 성 탄소의 정량 및 화합물 특정 동위 원소 분석 (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

식물 조직에서 유기산을 정량화하는 모세관 전기 영동을 사용하여 : 테스트 케이스 검사<em&gt; 커피 나무 아라비카</em&gt; 씨앗

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish between enantiomers (stereoisomers) of simple chiral species on the level of individual molecules. In this approach, a gaseous molecular jet of the sample expands into a vacuum chamber and intersects with femtosecond (fs) laser pulses. The high intensity of the pulses leads to fast multiple ionization, igniting a so-called Coulomb Explosion that produces several cationic (positively charged) fragments. An electrostatic field guides these cations onto time- and position-sensitive detectors. Similar to a time-of-flight mass spectrometer, the arrival time of each ion yields information on its mass. As a surplus, the electrostatic field is adjusted in a way that the emission direction and the kinetic energy after fragmentation lead to variations in the time-of-flight and in the impact position on the detector.
Each ion impact creates an electronic signal in the detector; this signal is treated by high-frequency electronics and recorded event by event by a computer. The registered data correspond to the impact times and positions. With these data, the energy and the emission direction of each fragment can be calculated. These values are related to structural properties of the molecule under investigation, i.e. to the bond lengths and relative positions of the atoms, allowing to determine molecule by molecule the handedness of simple chiral species and other isomeric features.

For small chiral species, Coulomb Explosion Imaging provides a new approach to determine the handedness of individual molecules.

Stereoisomers 사이 구별 하는 도구로 쿨롱 폭발 영상

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...