Ubp1을 표현 플라스미드 pCG001께서는 의학 연구의 존 커틴 대학에서 로한은 베이커에 의해 제공되었다. 이 작품은 천 연구 기금 1 FY2011 년 3 월, 천​​ 바질 오코너 초보자 학술 연구 상 # 5 - FY20 및 NSF 경력 상 0,746,796 3 월에 자금을 지원했다.

1. 스트레인 엔지니어링 워크 플로우 <ol><li> 시퀀스 인코딩 (A) 막 현지화 순서, (b)는 빠른 속도로 성장하고 형광 단백질과와 관심 단백질 (예를 들어 전사 인자)와 프레임 (C) 단백 분해 효소 인식 서열 N-터미널.를 삽입 우리는 TSR-비너스 기자 대상 멤브레인을 사용하고 표현 박테리오파지 λ 억제 CI 단백질 분자의 수를 계산하기 위해 Ubiquitin 단백 분해 효소 인식 서열 (UB)에 융합. 우리가 지역을 코딩 CI 야생 형을 대체하고 E.에 구조를 통합, 융합 단백질 TSR-비너스-UB-CI를 구축 방법에 대한 자세한 내용은 λ 레드 재조합 3을 사용 대장균의 염색체가, 심판에 자세히 설명되어 있습니다. 1. </li><li> 시퀀싱하여 모든 수정 사항을 확인합니다. </li><li> 플라스미드 인코딩 할 관심의 형광 기자와 단백질을 표현 변종에 위의 사용 된 인식 순서에 맞는 프로테아제를 변환병진 융합 인치 우리는 C-터미널 Ubiquitin 잔류 네 직후 cleaves 단백 분해 효소 Ubp1을 사용 하였다. </li></ol> 2. 문화 셀 및 샘플 준비 <ol><li> 한 E. 선택 갓 줄무늬 한천 플레이트에서 관심 대장균 식민지 하나 ML M9 최소한의 미디어 5로는 1X 가상 아미노산과 적절한 항생제로 보충. 원하는 온도로 흔드는에 품다는 충분히 달성 할 수 OD 600&gt; 1.0 (600 nm의에서 광학 밀도).에게 </li><li> OD 600 = 0.02에 해당 항생제로 1 ML 신선한 M9 매체에 문화를 희석. 적절한 온도에서 흔드는에 품다. 저온 성장에 필요한 경우 초기 세포 밀도 증가 될 수있다. </li><li> OD 600 = 0.2-0.3 (37에서 OD 600에서 초기 세포 배양과 ° C = 0.02, 이건 3-4 시간을 소요됩니다), 아가로 오스 겔 패드를 (3 단계) 준비를 시작합니다. </li><li> 때 OD 셀 이미징을위한 준비 <sub&gt;을 600 = 0.3-0.4. </li><li> 1.5 ML의 microcentrifuge 튜브, benchtop microcentrifuge에서 1 분 10,000 XG에서 원심 분리기로 한 ML incubated 문화를 전송합니다. </li><li> 표면에 뜨는을 취소하고 1 ML 신선한 M9 매체를 추가하고 펠렛을 부드럽게 resuspend. </li><li> 1 분에 10,000 XG에 원심 분리기. </li><li> 1.6 1.7 단계를 반복합니다. 표면에 뜨는 폐기하십시오. </li><li> 부드럽게 한 ML M9 미디어에 resuspend. timelapse 영상에 대한 낮은 세포 밀도를 보장하기 위해 100 배에 - 세포를 직접 현미경 이미징 또는 희석 10 사용할 수 있습니다. 낮은 세포 밀도가 확장 timelapse 영상을 위해 중요합니다. 이미징 챔버 (4 단계)은 실험이 진행되는 동안 봉인되어 있습니다. 세포의 기하 급수적 인 성장으로 인해, 높은 초기 세포 농도는 크게 형광 단백질 성숙을 줄이고 세포의 성장에 영향을 미치는, 젤 패드의 장기 성장 후 챔버 내에 산소를 고갈 할 수 있습니다. </li></ol> 3. 아가로 오스 솔루션을 준비 <ol><li> 10-20 MG를 달다1.5 ML의 microcentrifuge 관에 낮은 녹는 온도 아가로 오스. </li><li> 3 % 아가로 오스 솔루션을 만들기 위해 항생제없이 적절한 볼륨 M9 최소한의 매체를 추가 할 수 있습니다. </li><li> 70시 30 분의 아가로 오스 솔루션을 가열 ° C, 솔루션은 완전히 녹아 균일 성을 확보하기 위해 반전 튜브를 용해 할 수 있습니다. 젤 패드는이 점 (4 단계)에서 부어 할 수 있습니다 또는 온도는 ° C 50 감소하고 나중에 사용하기 위해 몇 시간 동안 실시 할 수 있습니다. </li></ol> 4. 상공 회의소에 젤 패드를 준비합니다 <ol><li> 이미징 전에 약 50 분, 물과 Microaqueduct 슬라이드를 씻어. </li><li> 하나는 커버 유리를 청소 (6에 설명 등 여러 엄격한 청소 방법을 사용하여)와 압축 공기로 불어하여 멸균 물과 건조로 슬라이드를 Microaqueduct 씻어. </li><li> 이렇게이의 응용 프로그램을 수정할 수 있습니다 Microaqueduct 슬라이드 (의 유리면에 유입구 및 유출구를 포함하도록 Microaqueduct 슬라이드에 고무 개스킷을 삽입합니다어떤 미디어)은 샘플을 통해 perfused 있습니다. Microaqueduct 슬라이드의 중심 50 μl 아가로 오스 솔루션 (3 단계)을 적용합니다. </li><li> 청소, 드라이 커버 유리와 아가로 오스 솔루션을 맨. </li><li> 아가로 오스 솔루션은 30 분 동안 실온에서 서 보자. </li><li> 한편, (제 2 단계) 세포 샘플을 준비합니다. </li><li> 조심스럽게 젤 패드의 상단에서 덮개 유리에서 껍질. 젤 패드의 상단에 1.0 μl 한물 세포 배양을 추가합니다. 젤 패드에 의해 흡수 될 수있는 문화 ~ 2 분 기다리십시오. 그건 너무 오래 기다리지 않고 중요하다 젤 패드 overdries하지만, 충분히 전지가 올바르게 젤 패드을 준수되었는지 확인합니다.주세요 이상적인 대기 시간은 온도와 습도에 따라 달라질 수 있습니다. </li><li> 기다리는 동안 다른 precleaned 커버 유리를 건조. </li><li> 커버 유리와 Microaqueduct 슬라이드가 잘 정렬되어 있도록 새로운 커버 유리로 샘플을 커버. </li><li> 제조업체의 지침에 따라 온도 제어 성장 챔버를 조립.그것은 지금 현미경 (단계 5)에서 영상을 위해 사용될 수 있습니다. </li></ol> 5. 이미징 및 Timelapse 영화의 취득 <ol><li> 제조업체의 지시 사항 (예 : 레이저 실험 전에 ~ 0.5 시간에 예열해야 할 수도 있습니다) 다음과 같은 레이저와 현미경을 사용합니다. </li><li> 현미경의 무대 삽입에 조립 챔버를 잠급니다. 성장 챔버의 온도와 37 ° C 나 기타 원하는 성장 온도에서 목표 히터를 설정합니다. </li><li> 실온 위 이미징 경우, 초점이나 젤 패드는 일반적으로 초기 온도 변화 후 ~ 15 분 동안 크게 표류합니다. 실제로, 관심 지역을 찾아 실험을 위해 필요에 따라 이미징 스크립트를 수정할 시간을 사용합니다. </li><li> 현미경에서 세포를 찾아 미리 정의와 정렬 이미징 지역 내에서 중심 한 후 각 셀의 위치를​​ 저장합니다. 하나 이상의 셀은 각 이미지 획득 시간 창에서 이미징 할 수 있습니다. m 이상 timelapse 영상에 대한모든 세대가 다른 식민지 성장하는 동안 이미징 지역을 입력하지 않도록 이미징 세포가 처음 백 회에 몇 μm하여 다른 세포로부터 분리되어 있는지 확인하십시오. </li><li> 그래서 레이저 여기 강도를 조정은 6 노출 내의 거의 모든 형광 분자의 photobleach (그림 5). </li><li> 자동 이미지 스크립트 / 잡지, 그림 3의 실험 워크 플로우에 설명 된 알고리즘을 사용하여 timelapse 데이터를 가져올을 사용합니다. </li></ol>

<ol>
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우리는 공개가 없습니다.

CoTrAC 방법은 기존 N 또는 C-터미널 형광 단백질 fusions 단백질의 활동을 방해하는 다른 단백질의 생산을 측정하는 일반화 할 수 있습니다. CoTrAC 전략은 현재의 방법으로 세 독특한 장점이 있습니다. 첫째, 공동 병진 융합 형광 기자 분자가 실시간으로 단백질 생산의 정확한 계산을 허용, 관심있는 단백질의 각 분자에 대한 생산 될 수 있도록합니다. 둘째, 막 타겟 기자는 TSR-금성은 매우 낮은 수준에?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 아가로 오스 (낮은 녹는 온도) </td><td> Lonza </td><td> 50,100 </td><td></td></tr><tr><td> 밀리 Q H 2 O </td><td></td><td></td><td></td></tr><tr><td> 5 × M9 소금 </td><td></td><td></td><td> 다음 제조법은 9에 설명 </td></tr><tr><td> 20 % 포도당 </td><td></td><td></td><td></td></tr><tr><td> MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> CaCl 2 </td><td></td><td></td><td></td></tr><tr><td> 50 × 가상 아미노산 솔루션 </td><td> Invitrogen </td><td> 11130-051 </td><td></td></tr><tr><td> 온도 조절 성장방 무대 어댑터 </td><td> Bioptechs </td><td> FCS-2 </td><td></td></tr><tr><td> 목표 히터 </td><td> Bioptechs </td><td> 모델 현미경 대물에 따라 달라집니다 </td><td></td></tr><tr><td> 슬라이드 Microaqueduct </td><td> Bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td> 마이크로 커버 유리 </td><td> VWR </td><td> 40CIR-1 </td><td> 소스에 어려울 수 있습니다, Bioptechs의 이용도 가능합니다 </td></tr><tr><td> 유리 / 슬라이드 근육 커버 </td><td> Bioptechs </td><td> FCS2 0.75 mm </td><td></td></tr><tr><td> 형광 현미경 </td><td> 여러 </td><td> 예를 들어 설정 : 코 히어 런트 Innova 308C 아르곤 이온 레이저, 올림푸스 IX-81 현미경, 올림푸스 PlanApo 100X NA 1.45 목적, Metamorph 소프트웨어 </td><td> 레이저 여기, 자동화 XYZ 단계, 스크립트 이미징 및 자동 초점이 가능 자동화 소프트웨어 resolvin 할 수있는 광학이 있어야합니다g 단일 형광 단백질 </td></tr><tr><td> EM-CCD 카메라 </td><td> 여러 </td><td> 예를 들어 설정 : ANDOR Ixon DU-898 </td><td> 배경 위의 하나의 형광 단백질을 감지 충분히 낮은 소음을 가지고 있어야 </td></tr></tbody></table>

single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)

우리는 이미지 절단하여 형광 현미경 방법, 공동 병진 인증 (CoTrAC) 단백질의 기능을 perturbing없이 단일 분자 정밀도 라이브 세포의 단백질 분자의 생산을 설명합니다. 이 방법은 가능한 연속 다섯 분 시간 창 동안 한 셀에서 생산되는 단백질 분자의 숫자를 계산 할 수 있습니다. 이 라이브 셀에 하나의 형광 단백질 분자를 감지 할 충분히 문자를 구분합니다 ~ 0.5-1 kW 급 / cm 2,의 레이저 여기 전력 밀도와 형광 현미경이 필요합니다. 막 타겟팅 순서, 빠른 속도로 성장하고, 노란색 형광 단백질과 단백 분해 효소 인식 순서 :이 방법에 사용되는 형광 기자는 세 부분으로 구성되어 있습니다. 기자는 translationally 관심 단백질의 N-말단에 융합되어 있습니다. 셀은 온도 조절 현미경 무대에서 성장하고 있습니다. 5 분마다, 세포 내 형광 분자 (이상 공동 이미징 아르) 형광 이미지를 분석하여 unted 만 새로 번역 된 단백질이 다음 측정에 포함됩니다 있도록 이후 photobleached. 이 방법에 의한 형광 이미지는 각 이미지의 형광 명소를 감지 개별 셀에 지정하고 세포 lineages에 셀을 지정하여 분석 할 수 있습니다. 주어진 셀에 시간 창 내에서 생산되는 단백질의 수는 단일 형광 분자의 평균 강도에 의해 장소의 통합 형광 강도를 나누어 계산됩니다. 우리는 하나의 5 분 시간 창에서 0-45 분자의 범위에서 표현 수준을 측정 할 수이 방법을 사용했습니다. 이 방법은 우리가 λ 억제 CI의 표현에 소음을 측정 할 수있게하고, 시스템 생물학에 많은 잠재적 인 응용 프로그램이 있습니다.

λ 억제 CI의 생산을 추적 CoTrAC 실험에서 전형적인 결과는 그림 4와 5에 표시됩니다. 이 실험에서, 12 콜로니는 5 분 간격으로 이미징했다. 때마다 시점에서, 식민지 먼저 autofocused되었고 이미징 영역 내에 중앙. 다음으로 중심 / 중심의 위치가 저장되었고 brightfield 이미지가 인수되었습니다. 무대는 다음 세포를 (위상 대비 또는 차등 간섭 대비 (DIC) 광학없이 bisecting 비행기에 brightfield 초?...

Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

우리는 이미지 절단하여 형광 현미경 방법, 공동 병진 인증 (CoTrAC), 단백질의 기능을 perturbing없이 단일 분자 정밀도 라이브 세포의 단백질 분자의 생산을 설명합니다. 이 메소드는 전사 인자, λ 억제 CI의 확률 표현 역학을 수행하는 데 사용되었습니다<sup&gt; 1</sup&gt;.

유전자 규정의 단일 분자 이미징<em&gt; 생체에</em&gt; 절단하여 Cotranslational 인증 (CoTrAC)를 사용

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

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JoVE Journal

Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

9.8K 조회수.  Johns Hopkins University School of Medicine. 우리는 이미지 절단하여 형광 현미경 방법, 공동 병진 인증 (CoTrAC), 단백질의 기능을 perturbing없이 단일 분자 정밀도 라이브 세포의 단백질 분자의 생산을 설명합니다. 이 메소드는 전사 인자, λ 억제 CI의 확률 표현 역학을 수행하는 데 사용되었습니다 1.

비디오: Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

원자력 교통 단일 분자 이미징

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

연구

생물학

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

시토크롬의 세포질 Microinjection 의한 Apoptosis의 활성화 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

절단 조각의 탐지와 전류 측정을 결합하여 상피 나트륨 채널 (ENAC)의 단백질 분해 활성의 데모

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

단일 분자 FRET에 의해 DNA 루핑을 공부

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

현장 별 DNA 분열을 위한 병렬 높은 처리량 단 분자 운동 분석

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...