This work was supported by the Lehigh University Start-up Package. We thank Dr. Walter Fast for providing the GST-PAD4 plasmid for protein expression.

1. PAD4 변환, 발현, 및 정제 <ol><li> PAD4 변환의 경우, 화학적으로 유능한 E. PAD4의 GST 융합을 포함하는 플라스미드를 pGEX 변환 다음 절차를 사용하여 단백질 발현 대장균 (BL21 (DE3)) 세포. 화학적 유능한 (염화 칼슘)를 준비 E. 표준 프로토콜에 따라 대장균 (BL21 (DE3)) 세포. <ol><li> 미리 제조 한 화학적 유능한 BL21 (DE3) 세포를 얼음에 50㎕의 해동 및 5 ㎖의 배양 관에 PAD4 유전자를 포함하는 플라스미드를 pGEX 1 μL와 혼합한다. 부드럽게 매 2 분 흔들어 섞으면 서 10 분 동안 얼음 혼합물을 품어. </li><li> 열은 40 초 동안 42 ° C의 물을 용기에 혼합물을 배치하여 셀을 충격. 즉시 세포가 복구 할 수 있도록 2 분 동안 다시 얼음에 세포 플라스미드 혼합물을 배치합니다. 혼합물에 멸균 된 LB 액체 배지 1 ㎖를 추가하고 1 분 동안 얼음에 배치합니다. </li><li> 1 250 rpm으로 흔들어, 37 ° C에서 세포 충격 열을 품어시간. 피펫 (75) 암피실린 내성 한천 접시에 형질 전환 세포의 μL, 15 시간 동안 37 ° C에서 품어. 4 ℃에서 보관 판. </li></ol></li><li> PAD4 식입니다. <ol><li> 1x를 앰피 실린을 함유하는 LB 5 ml의 암피실린 한천 플레이트와 장소에서 BL21 (DE3) 세포의 콜로니 하나를 선택. 인큐베이터에 넣어 37 ℃에서 O / N를 흔들. </li><li> 1x를 암피실린 수화물을 함유하는 멸균 LB (MW 403.45 g / mol)를 1 L로 LB (스타터)의 5 ml의 전송. 37 ° C 진탕 배양기에 넣어 성장. 성장의 OD (600)를 모니터링합니다. 성장 (600) 0.3의 OD에 도달하면, 인큐베이터을 흔들어 16 ° C로 성장 이동합니다. </li><li> OD 0.6 600 0.3 밀리미터 isopropylthiagalactoside (IPTG, MW 238.30 g / mol)을 가진 세포를 유도 도달하면. 세포가 16 ℃에서 15 시간 동안 흔들 수 있습니다. </li><li> 0 ° C에서 20 분 동안 4,000 XG에서 원심 분리하여 수확 세포. -80 ° C에서 상등액 및 저장 펠렛을 붓고. </li></ol></li><li> PAD4 정화 <ol><li> 다시 일시의 50 mM의 NaCl (MW 58.44 g / 몰)의 버퍼에 세포를 BL21 (DE3)에서 발현 PAD4 함유 펠렛을 300 mM의의 NaH 2 PO 4 (MW 119.98 g / mol)을 10 mM의 이미 다졸 (MW 68.077 g / 몰), 0.1 mM의 페닐 메틸 설 포닐 플루오 라이드 (PMSF, MW 174.94 g / mol) 및 1 mM의 디티 오 트레이 톨 (DTT, 154.25 g / 몰)의 pH = 8.0. </li><li> 4 ° C에서 15 분 동안 초음파 처리를 통해를 Lyse은 세포. 초음파 처리 후, 0 ° C에서 20 분 동안 20,000 XG에서 세포 용 해물을 원심 분리. 붓고 및 상층 액을 저장합니다. 일괄 RT에서 30 분 동안 글루타치온 (GSH), 아가 로스 비드와 상청. </li><li> 중력을 통해 GST 비즈 / 열에서 상층 액을 배출합니다. 실온에서 1X PBS (인산염 완충 식염수, pH는 8.0)의 4 × 25 ml로 구슬을 씻으십시오. </li><li> 용출 PAD4 2 × 10 ml의 용출 버퍼와, 50 mM의 트리스 (히드 록시 메틸) 아미노 메탄 (트리스 자료, MW 121.14 g / mol)을 10 mM의 글루타치온 (GSH, MW 307.32 g / 몰), pH가 8.0. </li><li> 10 만 MW 컷-O를 사용하여 PAD4을 집중FF 원심 분리기 튜브와 원심 분리기 4 ° C에서 20 분 동안 4,000 XG에. -80 ° C에서 나누어지는 1.0 ml의 마이크로 원심 튜브에 200 μL의 볼륨으로 단백질 및 저장. </li></ol></li></ol> 버퍼 2. 준비 원액 <ol><li> 염화나트륨 (NaCl을, MW 58.44 g / 몰) 칭량하고 2 M 용액을 제조 하였다. 명확 때까지 솔루션을 섞는다. </li><li> 트리스 (히드 록시 메틸) 아미노 메탄 (트리스 자료, MW 121.14 g / 몰) 밖으로 달아 = 8.0 2 M 용액의 pH를 준비합니다. 명확 때까지 솔루션을 섞는다. </li><li> 염화칼슘 이수화 물 (CaCl2를 2H 2 O, MW 147.01 g / 몰) 칭량하고 500 mM의 용액을 조제. 명확 때까지 솔루션을 섞는다. </li><li> 트리스 (2- 카르복시 에틸) 포스 핀 (TCEP, MW 250.19 g / 몰) 칭량하고 200 mM의 용액을 조제. -20 ° C에서 명확하고 저장까지 솔루션을 섞는다. </li><li> 디티 오 트레이 톨 (DTT, MW 154.25 g / 몰) 밖으로 무게와 1 M 용액을 제조. -20 ° C에서 명확하고 저장까지 솔루션을 섞는다. </li><li> 준비트리톤 X-100의 0.5 % 용액. </li><li> 에틸렌 디아민 테트라 아세트산 (EDTA, MW 292.24 g / 몰) 칭량하고 100 mM의 용액을 조제. 명확 때까지 솔루션을 섞는다. </li><li> 염산염 (ZRcoum, MW 621.69 g / mol)의 methylcoumarin Z- Arg- Arg- 7- amido- 4-을 달아 디메틸 설폭 사이드 (DMSO)에 10 mM의 용액을 조제. </li></ol> 37 ° C에서 3 PAD4 분석 <ol><li> 10 mM의 ZRcoum 재고에서 물에 ZRcoum의 125 μm의 솔루션을 준비합니다. 이 125 μM ZRcoum 솔루션은 솔루션 A. 될 것입니다 </li><li> 62.5 mM의 NaCl을, 62.5 mM 트리스, 12.5 mM의 CaCl2를, 6.25 mM의 DTT의 버퍼, 5 μM의 PAD4 (PH = 8.0)를 준비합니다. 이 솔루션 B. 될 것입니다 </li><li> 62.5 mM의 NaCl을, 62.5 mM 트리스, 12.5 mM의 염화칼슘 2, 6.25 mM의 DTT (PH = 8.0)의 버퍼를 준비합니다. 이 솔루션 C. 될 것입니다 </li><li> (소의 췌장에서) 트립신, 결정을 달아 100 mM의 EDTA에서 10 ㎎ / ㎖를 준비합니다. 명확 때까지 솔루션을 혼합하고 -20 ℃에 저장° C. 이 솔루션 D.됩니다 </li><li> 96 웰 플레이트를 확보하고 컨트롤 우물의 수 (NO PAD4) 및 테스트 우물 (+ PAD4)를 지정합니다. </li><li> 피펫 테스트 우물과 피펫 제어 우물에 솔루션 C의 160 μL에 솔루션 B의 160 μL. 37 ° C에서 15 분 동안 품어. </li><li> 피펫 (40) 모든 우물에 솔루션의 μL와 45 분 동안 37 ℃에서 배양한다. </li><li> 테스트 우물의 제어 우물의 절반과 절반 dH보다 2 O의 피펫 10 μL. 대조군 웰 및 테스트 제어, 피펫 37 ° C에서 10 분 동안 인큐베이션 D. 용액 10 μL의 다른 절반. </li><li> 345 나노 미터의 여기 파장 465 나노 미터의 발광 파장 복합 리더기에 읽어 형광을 얻습니다. </li></ol> RT 4. PAD4 분석 <ol><li> 10 mM의 ZRcoum 재고에서 물에 ZRcoum의 50 μm의 솔루션을 준비합니다. 이 50 μM의 ZRcoum 솔루션은 솔루션 A. 될 것입니다 </li><li> 100 mM의 나 버퍼를 준비CL, 100 mM 트리스, 20 mM의 CaCl2를, 2 mM의 TCEP, 0.02 % 트리톤 X-100, 8 μM의 PAD4 (pH가 = 8.0). 이 솔루션 B. 될 것입니다 </li><li> 100 mM의 NaCl을, 100 mM 트리스, 20 mM의 CaCl2를, 2 mM의 TCEP, 0.02 % 트리톤 X-100 (하여 pH = 8.0)의 버퍼를 준비한다. 이 솔루션 C. 될 것입니다 </li><li> (소의 췌장에서) 트립신, 결정을 달아 100 mM의 EDTA에서 10 ㎎ / ㎖를 준비합니다. -20 ° C에서 명확하고 저장까지 솔루션을 섞는다. 이 솔루션 D.됩니다 </li><li> 384 웰 플레이트를 취득 및 제어 (NO PAD4) 및 테스트 우물 (+ PAD4)에 대한 우물을 지정합니다. </li><li> 피펫 테스트 우물과 피펫 제어 우물에 솔루션 C의 25 μL에 솔루션 B의 25 μL. 실온에서 20 분 동안 우물에서 부화 PAD4 / 없음 버퍼를 허용합니다. </li><li> 모든 우물에 솔루션 C의 피펫 25 μL와 실온에서 45 분 동안 품어. </li><li> 테스트 우물의 제어 우물의 절반과 절반 dH보다 2 O의 피펫 10 μL. 대조군 웰 및 테스트 제어의 다른 절반에 pipett실온에서 10 분 동안 솔루션 D. 품어의 전자 10 μL. </li><li> 345 나노 미터의 여기 파장 465 나노 미터의 발광 파장 복합 리더기에 읽어 형광을 얻습니다. </li></ol> 37 ° C 및 실온에서 5 PAD4 시간 코스 분석 실험 <ol><li> 10 mM의 ZRcoum 재고에서 물에 ZRcoum의 125 μm의 솔루션을 준비합니다. 이 125 μM ZRcoum 솔루션은 솔루션 A. 될 것입니다 </li><li> 62.5 mM의 NaCl을, 62.5 mM 트리스, 12.5 mM의 CaCl2를, 6.25 mM의 DTT의 버퍼, 5 μM의 PAD4 (PH = 8.0)를 준비합니다. 이 솔루션 B. 될 것입니다 </li><li> (소의 췌장에서) 트립신, 결정을 달아 100 mM의 EDTA에서 10 ㎎ / ㎖를 준비합니다. -20 ° C에서 명확하고 저장까지 솔루션을 섞는다. 이 솔루션 C. 될 것입니다 </li><li> (얼마나 많은 시점에 따라 달라집니다 우물 #)이 96 웰 플레이트 및 피펫 우물 # 금액에 솔루션 B의 160 μl를 얻습니다. </li><li> 37 ° C에서 한 96 웰 플레이트를 품어. RT에서 다른 96 웰 플레이트를 품어. </li><li> 삐악 삐악 울다ette 즉시 (40) 모두 96 웰 플레이트의 모든 우물에 솔루션의 μL과는 t = 0 분 우물에 솔루션 C의 10 μl를 피펫. 모든 우물이 침묵 할 때까지 특정 시점에서 솔루션 C의 10 μl를 계속 추가합니다. </li><li> 345 나노 미터의 여기 파장 465 나노 미터의 발광 파장 복합 리더기에 읽어 형광을 얻습니다. </li></ol> 실온에서 6 PAD4 저해 분석 <ol><li> 10 mM의 ZRcoum 재고에서 물에 ZRcoum의 83 μm의 솔루션을 준비합니다. 이 83 μM의 ZRcoum 솔루션은 솔루션 A. 될 것입니다 </li><li> 된 C1-아미 딘 (MW 424.8 g / 몰) 칭량하고 100 μM 스톡 용액을 준비한다. 이 솔루션 B. 될 것입니다 </li><li> 100 mM의 NaCl을, 100 mM 트리스, 20 mM의 CaCl2를, 2 mM의 TCEP, 0.02 % 트리톤 X-100의 완충액, 8 μM의 PAD4 (하여 pH = 8.0)을 준비한다. 이 솔루션 C. 될 것입니다 </li><li> 100 mM의 NaCl을, 100 mM 트리스, 20 mM의 CaCl2를, 2 mM의 TCEP, 0.02 % 트리톤 X-100 (하여 pH = 8.0)의 버퍼를 준비한다. 목솔루션 D. 될 것입니다 </li><li> (소의 췌장에서) 트립신, 결정을 달아 100 mM의 EDTA에서 10 ㎎ / ㎖를 준비합니다. -20 ° C에서 명확하고 저장까지 솔루션을 섞는다. 이 솔루션 E. 될 것입니다 </li><li> 384 웰 플레이트를 확보하고 컨트롤 우물 (NO PAD4), 시험 우물 (+ PAD4) 및 억제 우물 (PAD4 + CL-아미 딘)을 지정합니다. </li><li> 피펫 테스트 우물과 피펫 제어 우물에 솔루션 D의 25 μL에 솔루션 C의 25 μL. 실온에서 20 분 동안 우물에서 부화 PAD4 / 없음 버퍼를 허용합니다. </li><li> 억제의 우물에 솔루션 B의 피펫 10 μL 및 RTE에서 20 분 동안 품어. </li><li> 모든 우물에 솔루션의 피펫 15 μL와 실온에서 45 분 동안 품어. </li><li> 피펫 모든 우물에 솔루션 E의 10 μL. 10 분 동안 RT에서 인큐베이션. </li><li> 345 나노 미터의 여기 파장 465 나노 미터의 발광 파장 복합 리더기에 읽어 형광을 얻습니다. </li></ol> 7. 원유 세포 용 해물 분석RT에서 <ol><li> 50 mM 트리스, 50 mM의 염화나트륨, 0.1 mM의 페닐 메틸 설 포닐 플루오 라이드 (PMSF, MW 174.94 g / mol) 및 1 mM의 디티 오 트레이 톨 (DTT, 154.25 g / mol)을, pH를 10 ㎖의 PAD4 1 L 성장에서 재현 탁 된 세포를 세포를 수확 한 후 = 8.0. </li><li> 4 ° C에서 15 분 동안 초음파 처리를 통해를 Lyse은 세포. 0 ° C에서 20 분 동안 20,000 XG에서 세포 용 해물을 원심 분리기. 10 만 MW 차단 원심 분리기 튜브를 사용하여 4 ° C에서 4,000 rpm으로 원심 분리하여 절반 볼륨에 맑은 상층 액을 집중한다. </li><li> 10 mM의 ZRcoum 재고에서 물에 ZRcoum의 250 μm의 솔루션을 준비합니다. 이 250 μM ZRcoum 솔루션은 솔루션 A. 될 것입니다 </li><li> 100 mM의 NaCl을, 100 mM 트리스, 20 mM의 CaCl2를, 2 mM의 TCEP (하여 pH = 8.0)의 버퍼를 준비한다. 이 솔루션 B. 될 것입니다 </li><li> (소의 췌장에서) 트립신, 결정을 달아 100 mM의 EDTA에서 10 ㎎ / ㎖를 준비합니다. -20 ° C에서 명확하고 저장까지 솔루션을 섞는다. 이 솔루션 C. 될 것입니다 </li><li> 블랙 384 웰 플레이트 및 설계를 얻컨트롤 (NO 세포 용 해물) 및 테스트 우물 (+ 세포 용 해물)에 대한 우물을 먹었다. </li><li> 피펫 시험의 우물에 모든 우물과 세포 용 해물의 피펫 60 μL에 B 액 100 ㎕. 실온에서 20 분 동안 품어. </li><li> 모든 우물에 솔루션의 피펫 40 μL와 실온에서 45 분 동안 품어. </li><li> 피펫 모든 우물에 솔루션 C의 10 μL. 10 분 동안 RT에서 인큐베이션. </li><li> 345 나노 미터의 여기 파장 465 나노 미터의 발광 파장 복합 리더기에 읽어 형광을 얻습니다. </li></ol>

<ol>
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The authors have nothing to disclose.

Herein, a fluorescence based assay was successfully developed that monitors the activity of PAD4. The assay has proven to be incredibly robust in a number of high-throughput conditions and is also compatible with whole cell lysates37.
The affinity between the histone proteins and the DNA strands surrounding it loosens due the neutralization of the positive charge on the arginine side chain upon citrullination. It was envisioned that the neutralization of the arginine side-chain coul...

포유 동물 단백질의 다수 무겁게 리보솜에 의한 단백질의 생합성 다음 효소의 작용에 의해 변성된다. 이러한 번역 후 변형 (PTMS)는 크게 단백질 1-3 (다른 기능 사이)의 크기, 전하, 구조 및 올리고머 상태를 변경하여 프로테옴의 기능적 다양성을 증가시킬 수있다. 그 결과, 단백질 구조의 변화는 단백질 분해, 세포 분화, 시그널링, 유전자 발현의 조절, 및 단백질 - 단백질 상호 작용과 같은 생리 학적 결과로 이어질 수있다. 이러한 수정은 인간의 모든 단백질의 큰 비율에 널리 퍼져 있지만 히스톤 단백질이 공유 수정 4의 비정상적으로 높은 숫자를 받아야에 터미널이 종료됩니다. 히스톤 단백질은 게놈 DNA의 응축을 촉진 구조 단백질의 패밀리이다. 구조화되지 않은 히스톤 꼬리의 공유 수정 세리 의해 수행 및 규제, 잔류 (작가)의 공유 변형을 촉진 같은 수정 (지우개), 역 및 변경을 구별 할 수 효소의 S는 히스톤 꼬리 (독자) 5-7에 각인된다. 실제로, 공지 PTMS 대부분 메틸화, 인산화, 아세틸, sumoylation, 유비퀴틴 및 citrullination 8 포함한 히스톤이 짧은 세그먼트 내에서 관찰 할 수있다. Citrullination는 비 t의 RNA 부호화 펩 티딜 - 시트룰린 (도 1a)에 펩 티딜 - 아르기닌의 변환을 수반한다. 이 측쇄 중화에 책임 단백질, PAD의 구성원 칼슘 의존적 효소이다 모두. 9,10이 단백질 군 (p는 rginine d를 eiminase eptide)를. 현재까지, 패드 가족의 다섯 멤버는 (PAD1, PAD2, PAD3, PAD4 및 PAD6)를 설명 하였다. 이 가족의 각 구성원은 별개의 세포 단백질을 대상으로 나타나고 또한 유니 표시가야 조직 분포 프로필. PAD4 핵 지역화 시퀀스 (11)를 통해 핵 내에서 지역화 된 것으로 알려진이 단백질 가족의 유일한 구성원입니다. 따라서,이 히스톤의 H2A 아르기닌 잔기 (3)의 N 말단 꼬리에서 아르기닌 측쇄 (H2R3), H3 (H3R2, H3R17 및 H3R26) 및 H4 (H4R3) (12)를 포함하여 핵 대상 번호를 deiminate 것으로 밝혀졌다, 13. PAD 동위 효소의 각 특정 중요한 생리 기능을 가지고 있지만, PAD4 인해 병에 걸린와 건강한 세포 모두에서 인간의 프로세스의 수의 역할에 훨씬 더 많은 관심을 받고있다. 최근 PAD4가 능성 전사 네트워크 (14)의 구성원이 될 것으로 나타났다. 두 PAD4 발현 수준과 활성 마우스에서 리 프로그래밍 및 접지 상태 능성 상태 동안 상승되는 것으로 도시 하였다. 줄기 세포 유전자의 조절을 제어함으로써, PAD4 세포 리 프로그래밍 효율의 중추적 인 역할을 유지할 수있다. PAD4도에 연루되었는지에 병원균의 결합 호중구 세포 트랩의 형성은 자신의 시스템 정리를 할 수 있습니다. PAD4 의한 히스톤 단백질의 hypercitrullination 따라서 세균 감염 15,16 떨어져 병동, 트랩에 의한 세포 외 병원균의 캡슐화 용 기재로서 기능 염색질의 decondensation을 유도한다. 또한 PAD4 인간 질병의 수가 활성 역할을하는 것으로 밝혀졌다. 것은 이미 PAD4의 비정상적 발현은 류마티스 관절염의 발병과 중증도와 연관되어 있음을 보여왔다, 알쯔하이머, ​​파킨슨 병 및 다발성 경화증 17. 실제로, 안티 - 시트룰린 단백질 항체의 존재는 류마티스 관절염 (18)의 가장 안전하고 확실한 진단 및 예후 바이오 마커이다. 마찬가지로, 조절 곤란 PAD4 활성은 최근 난소, 유방, 폐, 포함한 인간 암의 다수 관찰되었다D 식도암 19-22. PAD4과 암 사이의 링크는 p53 종양 억제 단백질 22, 23 ELK1 종양 유전자를 통해 또는 통해 매개되는 것으로 나타났다 이전 작업은 PAD4가 새로운 항암 치료 표적 17,24,25이 될 수 있다고 제안했다. 원리 연구의 증거로, 대장 암 세포주 HCT116에 shRNA를 통해 PAD4의 고갈은 세포 사멸과 세포주기 정지 (26)을 유도하기에 충분한 것으로 밝혀졌다. 최근에 개발 돌이킬 수 PAD4 억제제 쥐 27 종양 질량의 70 % 감소되었다. 아주 현저하게, PAD4 억제는 변형되지 않은 세포를 살려주는 동안 암 세포를 선택적으로 죽이는 결과 표적 치료의 역할을 한 것으로 보입니다. PAD4의 기능을 해제하는 작은 분자의 사용은 암 세포를 대상으로 또는 기존의 암 화학 요법 (28)을 증가시키기 위해 강력하고 새로운 전략으로 증명할 수 있습니다. 불행하게도, 포십t 가역 PAD4 억제제는 아직 발견해야합니다. 공유 억제제의 수는 아르기닌 기판 27,29,30 모방 건강 및 질병 상태 모두에서 세포 PAD4의 역할을 이해하는데 실질적인 도구적일 수 클로로 / 플루오 imidine 핸들을 사용하여 개발되어왔다. 그러나, 이들 분자는 유사한 효능을 가진 활성 패드 모두 억제한다. 따라서 PAD4의 활성에 대해보고 용이 한 분석에 대한 필요성이 매우 중요하다. 현재까지 PAD4 분석법 비색 판독 31 반응으로부터 암모니아의 방출을 연결하는 설명되었지만, 형광 편광 분석법 (32) 형광 표지 chloroamidine 기질 유사체를 이용하는 글리 옥살과 시트룰린 (33) 사이의 산 - 보조 반응에 의존하고, 몇 단계 34 dequenching 형광에 PAD4 활동. 이들 중에서, 단지 공유 개질제 haloacetamidine 전략은 높은 처리량 scre와 호환되도록 입증개로 인한 플랫폼 32,35,36. 우리는 확실 PAD4의 활성을 측정 손쉬운 형광 계 분석을 설명한다. 강한 신호 대 잡음비, 분석 속도 및 측정의 견고성을 표시 분석은, 진정으로 강력하고 선택 PAD4 억제제를 발견 할 가능성이있다.

<table border="0" cellpadding="0" cellspacing="0" width="716">
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			<td height="46" style="height:46px;width:192px;">Name of the Material/Equipment</td>
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			<td height="66" style="height:66px;width:192px;">LB Broth, Miller (LURIA-BERTANI)</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:135px;">J106-2KG</td>
			<td style="width:267px;"><a href="http://www.amresco-inc.com/">http://www.amresco-inc.com</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:192px;">Ampicillin Trihydrate (Off-white Powder), Fisher BioReagents</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td>BP902-25&#160;</td>
			<td style="width:267px;"><a href="http://www.fishersci.com/">http://www.fishersci.com</a></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:192px;">Isopropylthiagalactonse (IPTG)</td>
			<td style="width:123px;">Life Technogolies</td>
			<td>15529-019</td>
			<td style="width:267px;"><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com</a></td>
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		<tr height="62">
			<td height="62" style="height:62px;width:192px;">All Buffer Salts&#160;</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Can purchase from Fisher Scieintifc; VWR; Acros; Sigma-Aldrich; etc.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:192px;">Protino Glutathione (GSH) agarose&#160;</td>
			<td style="width:123px;">Machery-Nagel</td>
			<td style="width:135px;">745500.10</td>
			<td style="width:267px;">Store at 4 &#176;C; http://www.mn-net.com/</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Z-&#8203;Arg-&#8203;Arg-&#8203;7-&#8203;amido-&#8203;4-&#8203;methylcoumarin hydrochloride (Zcoum)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td>C5429</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:192px;">Chloroamidine&#160;</td>
			<td style="width:123px;">Cayman Chemical&#160;</td>
			<td style="width:135px;">10599</td>
			<td style="width:267px;">Store at -20 &#176;C; https://www.caymanchem.com/app/template/Home.vm</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:192px;">Tris(2-&#8203;carboxyethyl)&#8203;phosphine hydrochloride (TCEP HCl)</td>
			<td style="width:123px;">Thermo Scientifc</td>
			<td style="width:135px;">20490</td>
			<td style="width:267px;"><a href="http://www.piercenet.com/">http://www.piercenet.com</a></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:192px;">Triton X-100</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:135px;">X100-100ML</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Corning/Nunclon MicroWell plates (96 and 384)</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Purchase from Corning; Sigma-Aldrich</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Tecan Infinite 200 / Tecan i-control microplate reader software</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.tecan.com/">www.tecan.com</a></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Europium Ex. 340/40 Em. 475/15 filter</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.perkinelmer.com/">http://www.perkinelmer.com</a></td>
		</tr>
	</tbody>
</table>

fluorescence-based monitoring of pad4 activity via a pro-fluorescence substrate analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

처음에, 그것은 ZRcoum는 96- 웰 플레이트 형식으로 PAD4의 활성에보고 할 수 있음을 입증 하였다. 웰은 상기 기판과 함께 ZRcoum PAD4의 존재 / 부재 하에서 배양 하였다. 15분의 475 nm의 필터 (도 2A) - 37 ° C에서 45 분의 인큐베이션 기간 후, 형광 40분의 340로 측정 하였다. 예상 한 바와 같이, 형광 레벨은 ZRcoum (또는 시트룰린 ZRcoum) 내의 형광 ?...

Watch this Scientific Journal Video about Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog at JoVE.com

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

프로 형광 기판 아날로그를 통해 PAD4 활동의 형광 기반의 모니터링

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

fluorescence-based-monitoring-pad4-activity-via-pro-fluorescence

Research

JoVE Journal

Chemistry

9.8K 조회수.  Lehigh University. PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

비디오: Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

형광 기반 얼음 비행기 선호도에 의해 부동액 단백질의 얼음 결합 평면 결정

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

연구

화학

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.

This article describes a rapid supercritical extraction method for fabricating silica aerogels. By utilizing a confined mold and hydraulic hot press, monolithic aerogels can be made in eight hours or less.

빠른 초 임계 추출 방법을 통해 실리카 에어로젤, 모노 리드 (monolith)를 준비

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

연속 흐름 처리를 사용하여 수행 반응의 실시간 모니터링 : 예를 들어 3 Acetylcoumarin의 제조

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

BOC-알라 - 알라 -의 ASP-S-BZL의 가수 분해 : 색채 특히 그랜 자임 B 단백질 분해 활동를 측정 분석

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

방법은 표면 산화와 환원을 통해 액체 금속의 표면 장력을 조작

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

헬러의 HDAC1 및 HDAC6의 동시 측정 셀 사용 하 여 UHPLC MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

제조 및 급속 한 초 임계 추출 통해 준비 촉매 Aerogels의 테스트

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

활성 표면 성장 메커니즘을 통해 기판 바인딩된 Au 나노 와이어의 합성

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

이기에 친 환 경적 Steglich 에스테 르 화를 통해 에스테 르의 합성

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

화합물 억제, IC50 값, 관절 활성 및 뉴클레오시드 리보하이드라제의 전세포 활성을 결정하기 위한 NMR 기반 활성 반응

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...