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 border="0" cellpadding="0" cellspacing="0" width="1054">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">E. coli BL21(DE3)</td>
			<td style="width:135px;">Novagen</td>
			<td style="width:119px;">70235</td>
			<td style="width:367px;">DE3 lysogen contains T7 polymerase upon IPTG induction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">pET23d</td>
			<td style="width:135px;">Novagen</td>
			<td style="width:119px;">69748-3</td>
			<td style="width:367px;">pET23d confers resistance to ampicillin; T7 promoter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">LB Broth, Low Salt (Lennox L Broth), Granulated</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">GL1703</td>
			<td style="width:367px;">Conventional LB Broth, High Salt, can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Ampicillin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">10419313</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">Incubator Shaker (Thermostated)</td>
			<td style="width:135px;">NBS</td>
			<td style="width:119px;">Excella E24&#160;</td>
			<td style="width:367px;">Any shaker incubator with temperature control can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG), &#62;=99%</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:119px;">BP1755</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Sodium acetate, anhydrous, &#62;99%</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">B4017</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Ethylenediaminetetraacetic acid (EDTA), Disodium salt, &#62;99%</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:119px;">AC327205000</td>
			<td style="width:367px;">Harmful if inhaled.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge (refrigerated)</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:119px;">5430</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge (refrigerated) rotor</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:119px;">5430/5430P</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge</td>
			<td style="width:135px;">Sorvall</td>
			<td style="width:119px;">RC5C</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge rotor</td>
			<td style="width:135px;">Sorvall</td>
			<td style="width:119px;">SLA-3000</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">Phenol solution, Saturated with 0.1 M citrate buffer, pH 4.3 &#177; 0.2</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4682</td>
			<td style="width:367px;">Acute toxicity (oral, dermal, inhalation), corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Ethanol absolute for analysis, 100% v/v</td>
			<td style="width:135px;">Merck Millipore</td>
			<td style="width:119px;">100983</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Sodium phosphate dibasic dihydrate, Na2HPO4, &#62;=99%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">71643</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Potassium dihydrogen phosphate, KH2PO4</td>
			<td style="width:135px;">Merck</td>
			<td style="width:119px;">48,730,250</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">HiLoad 16/60 Superdex 75&#160;</td>
			<td style="width:135px;">GE Healthcare</td>
			<td style="width:119px;">28989333</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Agarose</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">MB1200</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Tris [Tris(hydroxymethyl) aminomethane HCl]</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">T1513</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Boric Acid</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">B0503</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Microwave</td>
			<td style="width:135px;">Haier</td>
			<td style="width:119px;">HDA-2070M</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">TCEP [Tris(2-carboxyethyl)phosphine hydrochloride]</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4706</td>
			<td style="width:367px;">Corrosive to metals and skin.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">ATP [Adenosine 5&#8242;-triphosphate disodium salt hydrate], Grade I, &#62;=99%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2383</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:433px;">Magnesium chloride (MgCl2), anhydrous, &#62;=98%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Sodium chloride (NaCl), &#62;99%</td>
			<td style="width:135px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP358</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Potassium chloride (KCl), &#62;99%</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">P0515</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">NZYDNA Ladder VI</td>
			<td style="width:135px;">NZYtech</td>
			<td style="width:119px;">MB8901</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Power supply</td>
			<td style="width:135px;">Consort</td>
			<td style="width:119px;">EV261</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Electrophoresis unit</td>
			<td style="width:135px;">Real Laboratory</td>
			<td style="width:119px;">RELSMINI10</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Real Safe nucleic acid staining</td>
			<td style="width:135px;">Real Laboratory</td>
			<td style="width:119px;">RBMSAFE</td>
			<td style="width:367px;">20,000X stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">UV Transilluminator (G:Box/EF)</td>
			<td style="width:135px;">Syngene</td>
			<td style="width:119px;">2216498</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Coomassie Brilliant Blue G</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0770</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Rocker (Gyro-Rocker)</td>
			<td style="width:135px;">Stuart</td>
			<td style="width:119px;">SSL3</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Mixer Vortex&#160;</td>
			<td style="width:135px;">Fisher brand</td>
			<td style="width:119px;">13214789</td>
			<td style="width:367px;"></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

Research

JoVE Journal

Biochemistry

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

9.7K 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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

효능 판별 분석에서 펩타이드 작용제의 직렬 희석에 대한 분배 자동 음향

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

연구

생화학

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

양 사체 (Transcriptome) 분석에있는 사용을위한 씨앗에서 매우 정제 된 RNA의 분리를위한 효율적인 방법

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

네이티브 전기 영동, In-gel Assay 및 Electroelution으로 미토콘드리아 전자 수송 사슬의 수퍼 콤플렉스 분석

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

2 차원 반 변성 세제 Agarose 젤 전기 이동 법 확인 또는 아 밀 로이드 같은 녹말 체 섬유의 크기가 하 사용

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

번역 개시 포유류 mRNAs에 복잡 한 대형의 Toeprinting 분석

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

안정적인 탄소 및 산소 동위 원소 분석을 위한 샘플링 및 치아에 나 멜의 전처리 탄산염

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

동적 Proteomic 그리고 미르 Langendorff 관류 후 절연 마우스 마음에서에서 Polysomes의 분석

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

블루 기본 젤 전기 이동 법에 의해 Thylakoid 막 단백질 복합물의 분석

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

선호도 크로마토그래피 수지의 신속한 개발을 위한 활성화된 상호 연결 아가로스 - 사례 연구로서의 항체 포획

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

시간 해결 된 단백질 유발 형광 향상을 활용하여 안정적인 지역 적합성을 식별하기 위해 한 번에 한 α 시뉴클레인 단백기를 식별합니다.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

단백질 분석을위한 Protein-tRNA Agarose Gel Retardation Assays

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