Name: protein-trna agarose gel retardation assays for the analysis of the n6-threonylcarbamoyladenosine tcda function
Uploaded: 2017-06-21T12:30:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function at JoVE.com

MCV는 CIB 교내 프로그램 &quot;더 나은 삶을위한 분자 기계&quot;(MACBET)의 회원입니다. 이 결과를 이끌어 낸 연구는 Salut Carlos III 스페인 연구소 (PI12 / 01667에서 MCV), 스페인의 Competitividad Ministerio Competitividad (CTQ2015-66206-C2-2-R 및 SAF2015-72961-MCV의 EXP)로부터 자금을 지원 받았다. ), 마드리드 지방 정부 (S2010 / BD-2316 ~ MCV) 및 유럽위원회 (Framework Program 7 (FP7)) 프로젝트 ComplexINC (계약 번호 279039 - MCV) 재단 기금은 연구 설계, 데이터 수집 및 분석, 출판 결정 또는 원고 작성에 아무런 역할을하지 못했습니다.

주의 : 사용하기 전에 관련된 모든 물질 안전 보건 자료 (MSDS)를 참조하십시오. 이 프로토콜에 사용 된 화학 물질 중 일부는 독성과 부식성이 있습니다. 흄 후드 및 개인 보호 장비 (안전 안경, 장갑, 실험실 코트, 긴 바지, 닫힌 발가락 신발 등 )의 사용을 포함하여 페놀을 사용하여 tRNA 추출을 수행 할 때 적절한 방법을 사용하십시오. 이 프로토콜은 무독성 핵산 얼룩을 사용합니다. 대체 얼룩의 사용은 독성 및 / 또는 발암 성 ( 예 : ethidium bromide)이있는 경우 염색제의 추가 예방 조치 및 특수 처분이 필요할 수 있습니다. 1. 아가로 오스 겔의 제조 <ol><li> 아가로 오스 2g을 달아 유리 병에 넣고 2 % w / v 아가 로스 겔을 만든다. </li><li> 아가로 오스 함유 병에 1X 트리스 - 보레이트 - 에틸렌 디아민 테트라 아세트산 (EDTA) (TBE) 러닝 버퍼 100 ML을 추가합니다. 전자 레인지에서 가열하여 녹입니다. 30 초 간격으로 내용물을 혼합하여 혼합하십시오아가로 오스가 완전히 용해 될 때까지이 단계를 반복하십시오. 아가 로스 용액을 약 50-60 ° C로 식힌다. </li><li> 겔 쟁반의 열린 가장자리를 테이프로 곰팡이를 만들고 적절한 빗을 넣어 우물을 만들고 용융 된 아가로 오스를 넣습니다. 거품 형성을 피하고 실온에서 응고 시키십시오. </li><li> 응고되면 겔 트레이를 전기 영동 장치에 넣고 겔이 완전히 덮일 때까지 TBE 완충액으로 채 웁니다. </li></ol> 2. tRNA의 제조 <ol><li> 대장균 에서 Express tRNA (UUU) <ol><li> 아침에 낮은 염 Luria Broth (LB)를 줄기 위해 발현 벡터 pET23d-EcUUU 2 ( 대장균 tRNA (UUU)를 코딩하는 유전자가 있음)로 형질 전환 된 냉동 E. coli BL21 (DE3) 100 μg / mL 암피실린을 보충 한 플레이트. 식민지가 나타날 때까지 접시를 뒤집고 37 ° C 배양기에 넣으십시오 (늦은 오후oon). </li><li> 5 ML LB + 100 μg / ML 암피실린에 잘 자란 단일 식민지를 접종하고 37 220 ML에서 밤새 성장 ° C. </li><li> 다음날 아침에 펠렛 세포 (4 ℃에서 10 분 동안 6,500 xg)를 5 ㎖ 신선한 LB + 100 ㎍ / mL 암피실린으로 재현 탁하고 재현 탁한다. 이 현탁액을 사용하여 200 mL LB와 100 μg / mL 암피실린을 접종하십시오. 37 ℃에서 220 rpm으로 5 시간 동안 성장시킨다. </li><li> 590 nm (OD 590 )에서 배양 물의 광학 밀도가 0.6 - 0.8에 도달 할 때, 1 mM IPTG를 배양 물에 첨가하여 tRNA (UUU) 유전자의 발현을 촉발시켰다. 37 ° C에서 3 시간 동안 배양하십시오. </li><li> 4 ° C에서 10 분 동안 6,500 xg에서 문화를 centrifuging하여 세포를 수확. </li></ol></li><li> 용해 및 추출 <ol><li> 5 ML의 용해 버퍼 (0.3 M 나트륨 아세테이트, 10 MM EDTA)에 세포 펠렛을 Resuspend. 이 시점부터 오토 클레이브 된 완충액을 사용하고 4 ℃에서 모든 tRNA- 함유 시료를 유지하여 tRNA 분해. </li><li> 흄 후드에 resuspended 세포 펠렛에 산성 페놀 (산도 4.3) 1 볼륨을 추가합니다. inversion에 의해 1 분간 혼합하여 세포를 용해시키고 10,000 xg (10 분, 4 ℃)에서 원심 분리한다. </li><li> 유기 (바닥) 단계를 대기음하지 않도록주의하면서 피펫을 사용하여 가용성 tRNA (UUU) (그리고 다른 tRNA 풀의 작은 농도)를 포함하는 상단 수성 단계를 수집하십시오. 유기층과 세포 잔해로 구성된 펠렛을 적절한 용기에 버리십시오. </li><li> 4 ° C에서 1 시간 동안 inversion하여 2.5 volume 100 % v / v 에탄올로 용해성 (수성) 상을 완전히 혼합하여 tRNA를 침전시킨다. 혼합물을 15,000 xg (30 분, 4 ° C)에서 원심 분리하십시오. </li><li> 뜨는을 조심스럽게 버리고 4 ML 겔 여과 버퍼 (20 MM 인산 나트륨 또는 인산 칼륨, 산도 7.4, 0.1 MM EDTA)에 tRNA가 풍부한 펠렛을 resuspend. 2 % w / v 아가로 오스 젤에 resuspended tRNA 풀 5 μL를 실행합니다. 노트:좋은 품질의 tRNA는 단일 밴드 (50-100 bp 사이의 분자 크기)로 나타납니다. </li></ol></li><li> tRNA (UUU)의 정제 <ol><li> 표준 크로마토 그래피 실습 12 에 따라 겔 여과 버퍼에서 미리 평형화 된 고해상도 분 취용 겔 여과 컬럼 (크기 : 16 x 600 mm, 베드 용적 : 120 mL, 분해능 범위 : 1,000 - 70,000 Da)에 재현 탁된 tRNA (UUU) (이동상). 샘플을 용리시키기 위해 유속을 1 mL / min으로 설정하십시오. 참고 : tRNA (UUU) 피크는 대략 75.5 mL에서 용리됩니다. 전형적으로, 배양 물의 0.5 내지 1.0 mg / L 수율의 tRNA가 수득 될 수있다. </li><li> 2 % w / v 아가로 오스 겔에서 각 시료 5 - 10 μL를 전기 영동하여 대표 용리 부피에서 시료를 분석하십시오. </li></ol></li></ol> 3. 샘플 준비 <ol><li> Lopez-Estepa et al .에 따라 TcdA를 표현하고 정제하십시오. 2 . 참고 : 최대 250 μMTcdA는 연구에 사용될 수 있습니다. </li><li> 표 1 에 따라 TcdA의 해당 양을 적절하게 표시된 1.5 mL 원심 분리 튜브에 피펫 팅합니다. 1.6 MM 트리스 (2-carboxyethyl) phosphine (TCEP) (최종 농도)을 추가하고 피펫을 사용하여 부드럽게 섞는다. 뚜껑을 닫고 5 분 동안 실온에서 혼합물을 품어. </li><li> 10 μM의 아데노신 트리 포스페이트 (ATP)와 50 mM MgCl 2 (최종 농도)를 첨가한다. 피펫과 혼합하고 실온에서 5 분 동안 혼합물을 품어. </li><li> 표 1에 따라 정제 tRNA (UUU) (섹션 2.3)를 추가하고 피펫과 섞은 다음 5 분 동안 실온에서 배양하십시오. </li><li> 최종 볼륨 100 μL까지 인산염 완충 식염수 (PBS)를 추가합니다. 제대로 혼합하고 10 % v / v의 최종 농도에 글리세롤을 첨가하십시오. </li><li> 시료를 보텍스하고 튜브의 내용물을 잠깐 스핀 다운하십시오 (10,000 xg, 1 분, 4 ° C). </li></ol> 4. 겔 적재 및 전기 영동 개발<ol><li> 전기 영동을 수행하는 동안 챔버를 4 ° C로 유지하기 위해 분쇄 얼음으로 채워진 트레이에 전기 영동 장치를 놓습니다. </li><li> 조심스럽게 각 샘플 5 μL를 해당 우물에 피펫 팅합니다. 적합한 DNA 크기 마커를 추가하십시오. </li><li> 전극을 전원 공급 장치의 해당 슬롯에 꽂습니다. 전원 공급 장치를 켜고 전압을 100V로 설정하고 90 분 동안 젤을 실행하십시오. </li></ol> 5. tRNA- 단백질 상호 작용을 관찰하기위한 젤 염색 <ol><li> TBE 50 ML과 적절한 DNA 얼룩 1X를 혼합하여 얼룩 솔루션을 준비합니다. 얼룩이있는 용기를 알루미늄 호일로 가려서 햇빛에 염색 용액을 노출시키지 마십시오. 참고 : DNA 얼룩은 일반적으로 10,000 또는 20,000 배 농축 주식으로 저장됩니다. </li><li> 전기 영동 90 분 후 조심스럽게 쟁반에서 젤을 제거하고 얼룩 솔루션과 컨테이너에 넣으십시오. 실내 온도에서 낮은 흔들림 속도로 로커에 올려 놓으십시오.30 분간 방치한다. </li><li> 염색 용기에서 아가로 오스 겔을 꺼내어 셀룰로오스 조직이나 여과지에주의하여 가볍게 두드려 과잉 액을 제거하고 직접 투광기 위에 놓습니다. 자외선을 켜고 tRNA가 포함 된 밴드를 시각화하십시오. 젤의 사진을 찍으십시오. </li><li> 단백질 염색 (선택). <ol><li> DNA 염색 용액을 안전하게 폐기하고 (안전 절차에 따라) 50 mL의 Coomassie Brilliant Blue (CBB) 용액으로 대체하십시오. 트레이를 실온에서 로커에 놓고 밤새 얼룩이 져야합니다 (&gt; 12 시간). 과도한 염료를 제거 CBB 얼룩 솔루션을 폐기하고 50 ML PBS로 교체하십시오. 로커에서 1 시간 동안 처치하십시오. </li></ol></li><li> 가벼운 transilluminator에서 단백질 함유 tRNA 복합체를 시각화하고 젤의 사진을 찍어 tRNA UV 젤 그림과 비교하십시오. </li></ol>

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저자는 공개 할 것이 없습니다.

본원에 기술 된 단백질 -tRNA 아가로 오스 겔 지연 분석법은 여러 가지 방법으로 변형 될 수있다. 첫째, 젤에서 아가로 오스의 비율은 여기 분석 (120 kDa)보다 훨씬 큰 단백질 - tRNA 복합체의 분리 및 시각화를 허용하기 위해 줄일 수 있습니다. 둘째, 목표 단백질이 열 안정성이있는 경우, 전기 영동 장치를 차가운 방으로 옮기거나 전기 영동 챔버 내부의 얼음 팩을 사용하여 생성 된 열을 즉시 소산시?...

<html> 겔 지연 분석 1 (전기 이동성 이동 분석, EMSA라고도 함)은 단백질 - 핵산 상호 작용을 연구하고 특성화하기 위해 고안된 전기 영동 방법입니다. 단백질 - RNA 상호 작용뿐 아니라 정제 된 성분 또는 단백질 ( 예 : 세포 용 해물) 또는 핵산 ( 예 : tRNA)의 복잡한 혼합물을 사용하여 tRNA와 tRNA 결합 단백질 간의 상호 작용뿐만 아니라 단백질 -DNA의 분석도 가능합니다 수영장). 우리는 정제 된 tRNA (UUU)와 anticodon stem loop (ASL)에서 A37에 부착 된 threonylcarbamoyl 측쇄를 고리 화하는 N 6 - threonylcarbamoyladenosine (t 6 A) 탈수 효소 인 TcdA 사이의 상호 작용 연구에 겔 지연 분석을 적용했습니다. tRNA의 cyclic t 6 (ct 6 A) 2 , 3 로의 전환. TcdA는 또한 CsdL 3 ,tcdA / csdL 유전자는 csdA 와 csdE 유전자를 갖는 클러스터를 형성하는데, 시스테인 탈황 효소와 시스테인 술핀 데스 탈 루핀 (CSD) 시스템의 황 수용체를 암호화하고 생체 내에서 TcdA 기능을 유지해야한다. 겔 지연 분석법의 근거는 유리 핵산 분자가 큰 음전하 때문에 아크릴 아마이드 또는 아가로 오스 겔의 앞쪽으로 빠르게 이동하지만 동일한 핵산의 단백질 복합체의 전기 영동 이동도가 크게 감소한다는 것입니다. 이동성의 감소는 핵산 - 단백질 복합체에서 &quot;이동&quot;으로 시각화되어 개별 밴드로 분해됩니다. 양전하를 띠고 큰 핵산 - 단백질 복합체는 겔상에서보다 큰 이동도 또는 이동도 감소를 나타낸다. 복합체의 전하 중화는 보편적 인 현상이므로단백질 - 핵산 복합체의 경우, 겔 지연 분석은 광범위한 복합체 및 핵산 유형에 적용될 수 있습니다. 이 방법은 간단하고 저렴하며 최소한의 장비로 실험실에서 수행 할 수 있습니다. 소량의 단백질과 tRNA 만 있으면됩니다. 이것은 대체로 더 많은 양의 표본을 소비하는 대체 생물 물리 기술보다 유리합니다. 겔 지연 분석은 전사 인자 연구에 널리 사용되었습니다. 이 분석법은 많은 다른 DNA 서열에 대한 전사 인자의 결합 동역학, 강도 및 특이성을 분석하는 데 사용되었습니다. 이 분야에서 실제로 EMSA가 처음 개발 된 것은 6 , 7 이었다. RNA 8 , 9 , 10 , 11 및 tRNA 분자를 이용한 겔 지연 분석도 리보솜 연구에 사용되었습니다우리가 여기에서와 같이 전사 후 tRNA 뉴 클레오 시드를 변형시키는 효소를 분석 할 수 있습니다. 다양한 매개 변수가 온도, 단백질 및 tRNA 샘플의 품질, 결합 강도와 같은 겔 지연 분석의 결과에 영향을 미칩니다. 실험의 신중한 계획과 실행은 유리 핵산과 단백질 결합 종의 결과 해석에 중요합니다. 여기에서, 프로모터가 Shine-Dalgarno 리보솜 결합 부위가 결여 된 발현 벡터에 클로닝 된 tRNA (UUU)를 코딩하는 합성 유전자를 사용하여 다량의 tRNA 2 의 합성을 유도 하였다. 이 상세한 프로토콜은 일반적인 실수를 피하면서 tRNA 겔 지연 실험을 수행하기위한 명확한 안내서를 제공하기위한 것입니다.

<table><tbody><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21(DE3)</td><td>Novagen</td><td>70235</td><td>DE3 lysogen contains T7 polymerase upon IPTG induction.</td></tr><tr><td>pET23d</td><td>Novagen</td><td>69748-3</td><td>pET23d confers resistance to ampicillin; T7 promoter.</td></tr><tr><td>LB Broth, Low Salt (Lennox L Broth), Granulated</td><td>Melford</td><td>GL1703</td><td>Conventional LB Broth, High Salt, can also be used.</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>10419313</td><td></td></tr><tr><td>Incubator Shaker (Thermostated)</td><td>NBS</td><td>Excella E24&amp;nbsp;</td><td>Any shaker incubator with temperature control can be used.</td></tr><tr><td>Isopropyl-beta-D-thiogalactopyranoside (IPTG), &gt;=99%</td><td>Acros Organics</td><td>BP1755</td><td></td></tr><tr><td>Sodium acetate, anhydrous, &gt;99%</td><td>Melford</td><td>B4017</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA), Disodium salt, &gt;99%</td><td>Acros Organics</td><td>AC327205000</td><td>Harmful if inhaled.</td></tr><tr><td>Centrifuge (refrigerated)</td><td>Eppendorf</td><td>5430</td><td></td></tr><tr><td>Centrifuge (refrigerated) rotor</td><td>Eppendorf</td><td>5430/5430P</td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall</td><td>RC5C</td><td></td></tr><tr><td>Centrifuge rotor</td><td>Sorvall</td><td>SLA-3000</td><td></td></tr><tr><td>Phenol solution, Saturated with 0.1 M citrate buffer, pH 4.3 &amp;plusmn; 0.2</td><td>Sigma-Aldrich</td><td>P4682</td><td>Acute toxicity (oral, dermal, inhalation), corrosive</td></tr><tr><td>Ethanol absolute for analysis, 100% v/v</td><td>Merck Millipore</td><td>100983</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, &gt;=99%</td><td>Sigma-Aldrich</td><td>71643</td><td></td></tr><tr><td>Potassium dihydrogen phosphate, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>48,730,250</td><td></td></tr><tr><td>HiLoad 16/60 Superdex 75&amp;nbsp;</td><td>GE Healthcare</td><td>28989333</td><td></td></tr><tr><td>Agarose</td><td>Melford</td><td>MB1200</td><td></td></tr><tr><td>Tris [Tris(hydroxymethyl) aminomethane HCl]</td><td>Melford</td><td>T1513</td><td></td></tr><tr><td>Boric Acid</td><td>Melford</td><td>B0503</td><td></td></tr><tr><td>Microwave</td><td>Haier</td><td>HDA-2070M</td><td></td></tr><tr><td>TCEP [Tris(2-carboxyethyl)phosphine hydrochloride]</td><td>Sigma-Aldrich</td><td>C4706</td><td>Corrosive to metals and skin.</td></tr><tr><td>ATP [Adenosine 5&amp;prime;-triphosphate disodium salt hydrate], Grade I, &gt;=99%</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;), anhydrous, &gt;=98%</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Sodium chloride (NaCl), &gt;99%</td><td>Fisher Bioreagents</td><td>BP358</td><td></td></tr><tr><td>Potassium chloride (KCl), &gt;99%</td><td>Melford</td><td>P0515</td><td></td></tr><tr><td>NZYDNA Ladder VI</td><td>NZYtech</td><td>MB8901</td><td></td></tr><tr><td>Power supply</td><td>Consort</td><td>EV261</td><td></td></tr><tr><td>Electrophoresis unit</td><td>Real Laboratory</td><td>RELSMINI10</td><td></td></tr><tr><td>Real Safe nucleic acid staining</td><td>Real Laboratory</td><td>RBMSAFE</td><td>20,000X stock</td></tr><tr><td>UV Transilluminator (G:Box/EF)</td><td>Syngene</td><td>2216498</td><td></td></tr><tr><td>Coomassie Brilliant Blue G</td><td>Sigma-Aldrich</td><td>B0770</td><td></td></tr><tr><td>Rocker (Gyro-Rocker)</td><td>Stuart</td><td>SSL3</td><td></td></tr><tr><td>Mixer Vortex&amp;nbsp;</td><td>Fisher brand</td><td>13214789</td><td></td></tr></tbody></table>

protein-trna agarose gel retardation assays for the analysis of the n6-threonylcarbamoyladenosine tcda function

우리는 Escherichia coli 에서 tRNA (UUU)의 발현과 정제를위한 방법과 tRNA (UUU)와 N6- threonylcarbamoyladenosine (t6 A) 탈수 효소 인 TcdA의 결합에 대한 겔 지연 분석에 의한 분석을 시연한다. threonylcarbamoyl cyclic t6 A (ct6 A)에 tRNA의 anticodon stem loop (ASL)에서 A37에 부착 된 측쇄. tRNA (UUU)를 암호화하는 합성 유전자의 전사는 1 ​​mM 이소 프로필 β-D-1-thiogalactopyranoside (IPTG)를 갖는 대장균 에서 유도되고 tRNA를 함유하는 세포는 유도 후 24 시간에 수확된다. RNA 분획은 산성 페놀 추출법을 사용하여 정제한다. 순수한 tRNA는 소형 tRNA 분자를 더 큰 손상되지 않은 핵산 또는 조각난 핵산에서 효율적으로 분리하는 겔 여과 크로마토 그래피에 의해 얻어집니다. tRNA (UUU)에 대한 TcdA 결합을 분석하기 위해 TcdA를 tRNA (UUU)와 혼합하고 4 ℃에서 천연 아가 로스 겔로 분리한다.TcdA-tRNA (UUU) 복합체는 젤의 염색시 관찰 할 수있는 이동성 지연을 겪는 반면, 자유 tRNA (UUU)는 더 빠르게 이동합니다. 우리는 TcdA가 tRNA (UUU) 결합 효소임을 입증합니다. 이 겔 지연 분석은 TcdA 돌연변이 체와 첨가제 및 기타 단백질이 결합에 미치는 영향을 연구하는 데 사용할 수 있습니다.

강력한 유도 성 T7 프로모터의 제어하에 대장균 에서 tRNA 유전자를 발현시킴으로써 대량의 tRNA (UUU)를 얻을 수있다. 발현 된 tRNA (UUU)는 세포질에 축적되며 자연적으로 풍부한 tRNA 풀을 풍부하게합니다. 포획 / 추출 단계 및 겔 여과 / 연마 단계로 이루어진 2 단계 정제 공정을 이용하여 EMSA 급 tRNA를 수득 하였다. 포획 / 추출 단계는 단백질, 세포 파편, DNA 및 tRNA (및 기?...

Watch this Scientific Journal Video about Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function at JoVE.com

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

프로토콜은 tRNA (UUU)의 생산 및 효소 TcdA와 복합체로 된 tRNA (UUU)의 분석을 아가로 오스 겔 지연 분석법을 통해 제시합니다.

단백질 분석을위한 Protein-tRNA Agarose Gel Retardation Assays<em&gt; N</em<sup&gt; 6</sup&gt; -threonylcarbamoyladenosine TcdA 기능

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

protein-trna-agarose-gel-retardation-assays-for-analysis-n6

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JoVE Journal

Biochemistry

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

9.8K 조회수.  Spanish National Research Council (CSIC).  프로토콜은 tRNA (UUU)의 생산 및 효소 TcdA와 복합체로 된 tRNA (UUU)의 분석을 아가로 오스 겔 지연 분석법을 통해 제시합니다. 

비디오: Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

준비 및<em&gt; 생체</em&gt; 액티비티 기반 프로브의 사용을위한<em&gt; N</em&gt; -acylethanolamine 산 아미 다제

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

연구

생화학

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Entamoeba histolytica DNA와 tRNA Methyltransferase Dnmt2 (Ehmeth) 효소를 체외 tRNA의 Methylation 분석에서

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

면역학 및 감염병학

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

인간의 Telomerase 역전사 단백질의 RNA 의존 RNA 중 합 효소 활동의 반 정량적 검출

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

유전학

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

효율적인 정화와 10-11 전-2 5-Methylcytosine Dioxygenase에 대 한 LC-MS/MS 기반 분석 결과 개발

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

TRNA Isopentenyl 전이 효소 활동을 감지 하는 체 외에서 분석 결과

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNA 메틸 트랜스퍼라제 활성 분석을 위한 항체 무첨가 분석

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

<em&gt; 체외</em&gt; 효소 분석은 갈륨 (III) 및 H에 의해 전사 억제를 측정하기 위해<sub&gt; 3</sub&gt; 5,10,15 트리스 (펜타 플루오로 페닐) corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

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Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

뉴 클레오 캡시드 소둔 매개 전기 영동 (NAME) 분석은 HIV-1 뉴 클레오 캡시드 억제제의 신속한 식별을 할 수 있습니다

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

DNA 단백질 상호 작용을 연구하는 CD 분광법

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

단백질 분석을위한 Protein-tRNA Agarose Gel Retardation Assays

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