이 작업은 FAPESP 보조금 (15/06477-9)에 의해 지원되었다.

1. 효모 총 추출물 준비
<ol><li>TAP태그9에 융합된 접합인자 중 하나를 발현하는 효모 세포를 성장시키는 데 적합한 아미노산 또는 핵염기와 함께 L YNB-glu 미디어(효모 질소 기준 2%m/v 포도당으로 보충됨), 이 경우 아데닌(20 μg/mL), 류신(30 μg/mL), 트립토판(30 μg/mL), OD600 = 1.0까지.</li>
	<li>1L 배양으로부터 100mL 원심분리기 병 3개에서 17,000x g에서 4°C에서 10분 동안 원심분리하여 세포를 수집하고 10mL 의 차가운 멸균물로 두 번 세척한다.</li>
	<li>수집된 효모 세포를 콜드 버퍼 A의 세포 부피 1/10에서 재연하여(10mmolL-1 HEPES pH = 7.9, 1.5mmol L-1 MgCl2,50mmolL-1 KCl, 5% v/v 글리세롤, 0.5mmolL-1 DTT, EDTA-프리 프로제스트 억제제).</li>
	<li>액체 질소에 셀 현탁액의 작은 방울을 동결. 작은 방울은 재중단 된 세포 용액을 파이프팅하고 50 μL을 액체 질소에 직접 떨어뜨려 얻을 수 있습니다. 주의: 액체 질소는 피부 또는 눈과 접촉하여 부상을 입을 수 있습니다. 적절한 안전 장비를 사용하여 처리하십시오. 참고: 프로토콜은 여기에서 일시 중지할 수 있습니다. 셀 서스펜션의 냉동 방울은 -80 °C에 저장 될 수있다.</li>
	<li>20Hz/s에서 6사이클을 통해 볼밀 장치에서 3분 동안 효모 세포용광을 준비합니다. 참고: 각 주기 후에, 녹지 않도록 액체 질소에 얼어붙은 세포를 품고 있는 용기를 담그세요. 프로토콜은 여기에서 일시 중지할 수 있습니다. 냉동 추출물은 -80°C에 보관할 수 있습니다. 효모 접합 추출물은 균질화제를 사용하여 구엽세포10을용해하거나 박격포 및유봉(11,12)을사용하여 제조할 수 있다.</li>
	<li>실내 온도에서 물에 들어 있는 튜브를 물에 넣고 가끔 흔들어 서 추출물을 녹입니다.</li>
	<li>원심분리기는 4°C에서 1h에 45,000 x g에서 추출합니다.</li>
	<li>BCA방법(13)에의해 지워진 상체의 단백질 함량을 정량화한다.</li>
	<li>추출물의 알리쿼트를 준비하고 액체 질소에 빠르게 고정하고 -80 °C에 보관하십시오. 참고: 프로토콜은 여기에서 일시 중지할 수 있습니다.</li>
</ol>2. 글리세롤 그라데이션 준비
<ol><li>버퍼 A에 두 개의 글리세롤 솔루션을 준비, 하나는 포함 10% v/v 와 다른 포함 30% v/v 글리세롤.</li>
	<li>30% v/v 글리세롤 용액에 교차 연결 제글루타랄데히드를 0.1% v/v에 넣고 혼합하여 균질화합니다. 주의: 적절한 안전 장비를 사용하여 연기 후드 내부의 글루타랄데히드를 처리하십시오.</li>
	<li>12mL 원심분리기 튜브(14 x 89mm) 하단에 차가운 10% v/v 글리세롤 용액 6mL를 추가합니다.</li>
	<li>튜브 하단에 그라데이션 마스터 장치가 제공된 긴 바늘에 부착된 주사기로 글루타랄데히드로 보충된 감기 30% v/v 글리세롤 용액6mL을 10% v/v 글리세롤 용액 아래에 추가합니다. 참고: 또는, 10% v/v 글리세롤 용액은 30% v/v 글리세롤/글루타랄데히드 용액의 상단에 조심스럽게 파이프화될 수 있다.</li>
	<li>그라데이션 마스터 장치를 사용하여 연속 밀도 그라데이션을 생성합니다. 참고: 그라데이션 마스터 장치에서 튜브는 매개 변수(시간/각도/속도)를 결정하기 위한 제조업체의 권장 사항에 따라 적절한 랙에 배치되고 잠시 회전됩니다. 이 작품에서 우리는 사용 : 2:25 분 / 81.5 ° / 11 rpm.</li>
	<li>셀 추출물을 첨가하기 직전에 7% v/v 글리세롤/버퍼 A 쿠션의 200 μL을 조심스럽게 상단에 넣습니다. 참고: 쿠션의 글리세롤 농도는 선형 그라데이션을 만드는 데 사용되는 농축 글리세롤 용액보다 낮어야 합니다.</li>
</ol>3. 원심분리추출
<ol><li>쿠션으로 12mL 선형 글리세롤 그라데이션 10%-30% 글루타랄데히드의 상단에 총 단백질 약 2 mg을 적재합니다.</li>
	<li>미리 냉각된 스윙 버킷 로터에 튜브를 놓습니다. 참고: 초원심분리 전에 혼합하지 않도록 튜브를 부드럽게 처리합니다.</li>
	<li>원심분리기는 4°C에서 16h의 194,000 x g에서 원심분리기.</li>
	<li>Aliquot각 12 mL 튜브24 500 μL 분획적응 된 EconoSystem을 사용 하거나 신중 하 게 파이펫팅에 의해 분수. 참고: 적응된 EconoSystem은 연동 펌프, UV 검출기 및 튜브 천포링 장치에 연결된 분수 수집기로 구성됩니다. 침전 프로파일은 280 nm에서 흡광도의 측정에 의해 모니터링될 수 있다.</li>
	<li>연동 펌프를 통해 40% v/v 글리세롤 용액을 사용하여 튜브에서 분획 수집기로 글리세롤10%-30%의 그라데이션을 밀어넣습니다. 참고: 프로토콜은 여기에서 일시 중지할 수 있습니다. 사용 전까지 분획을 -80°C에 보관합니다.</li>
	<li>점 얼룩 또는 슬롯 블롯 장치에 니트로 셀룰로오스 멤브레인에 직접 각 분획의 50 μL을 로드합니다. CBP에 대한 항체를 이용한 면역블롯(1:6,000, TAP 태그의 CBP 부분을 검출함) 및 호스레디시어 페록시다제(1:15,000)와 결합한 항래빗 IgG를 이차 항체로 검출한다.</li>
</ol>

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저자는 이해 상충이 없습니다.

단백질 단백질 및 리보핵산 단백질 상호 작용은 교차 연결제를 사용하여 안정화될 수 있다. 결과 복합체는 글리세롤 그라데이션에 대한 초원심분리를 견딜 수 있는 안정성이 있는 것이 중요합니다. 또한 버퍼 조건은 상호 작용을 허용해야하지만 비특정 바인딩을 피할 수 있을 만큼 엄격해야 합니다. 여기에 표시된 실험에서, 우리는 이미 시험관 내 접합 반응(15)에대해 확립 된 ?...

접합은 조정된 방식으로 다양한 요인의 바인딩 및 방출이 필요한 매우 역동적인 프로세스입니다. 이러한 접합 요인은 RNA 결합 단백질, ATPases, 헬리케이스, 단백질 키나아제 및 인산염, 유비퀴틴 리개세, 그 외1,2,3을포함한다. 그리고 분자 재배열이 일어날 수 있도록, 이러한 요인 중 일부는 매우 일시적인 이 RNP 중간 복합체의 격리 및 식별을 매우 어렵게 만듭니다.
여기서, 그라픽스 법4,5는 B액트 복합체 6과 효모 접합인자 Cwc24의 상호작용을 안정화하고, 유비퀴틴 리구아제 Prp19가 19(NTC)의 결합 또는 방출에 어떤 역할을 하는지 여부를 결정하기 위해B액트 복합체6을 사용하였다. 장소. 글리세롤 그라데이션을 따라 크로스링커의 농도가 증가하는 거시분자를 노출시키는 장점은 복합간 교차링크4,5,따라서 골재형성을 피한다는 것이다.
이 방법은 단백질 면역 침전 및 풀다운 분석에 대한 보완으로 사용되었으며, 이는 큰 복합체의 분리를 허용함에도 불구하고 큰 동적복합체7,8내에서 과도 상호 작용을 유지하는 데 신뢰할 수 없을 수 있다. 글리세롤 그라데이션에서 고정 시약을 사용하면 이러한 요인의 결합을 안정화시켜 접합 서브콤플렉스와 특정 단백질의 상호 작용을 확인할 수 있습니다. 선택된 크로스링커는 화학적으로 돌이킬 수 없기 때문에, 회수된 분획에 존재하는 단백질은 그라데이션 원심분리 후 점블롯에 의해 분석되었다.

<table>
	<tbody>
		<tr>
			<td>Anti-Calmodulin Binding Protein Epitope</td>
			<td>Millipore</td>
			<td>07-482</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-Rabbit IgG</td>
			<td>GE Healthcare</td>
			<td>NA934</td>
			<td></td>
		</tr>
		<tr>
			<td>EconoSystem</td>
			<td>Bio-Rad</td>
			<td>1-800-424-6723</td>
			<td>Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector</td>
		</tr>
		<tr>
			<td>EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Recovery System</td>
			<td>Beckman Coulter</td>
			<td>270-331580</td>
			<td>Tube-perforating device that was connected to the parts of the EconoSystem</td>
		</tr>
		<tr>
			<td>Gradient Master Model 107ip</td>
			<td>Biocomp</td>
			<td>107-201M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Mill MM 200</td>
			<td>Retsch</td>
			<td>207460001</td>
			<td>Ball Mill device</td>
		</tr>
		<tr>
			<td>Rotor F12-6x500Lex</td>
			<td>Thermo Scientific</td>
			<td>096-062375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall RC 6 Plus Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>36-101-0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging Bucket Rotor P40ST</td>
			<td>Hitachi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge CP 80 NX</td>
			<td>Hitachi</td>
			<td>901069</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear Centrifuge Tubes (14 x 89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes

pre-mRNA 접합은 복잡한 성분의 조립, RNA 처리 및 방출 중에 화려한 하위 복합체의 많은 분자 재배열을 포함하는 매우 역동적인 과정입니다. 글리세롤 그라데이션 원심분리는 기능적 및 구조적 연구를 위해 단백질 또는 RNP(RiboNucleoProtein) 복합체의 분리를 위해 사용되어 왔다. 여기서는 단일 입자 저온 전자 현미경 검사법을 위한 거대 분자 복합체를 정화하고 안정화하기 위해 처음 개발된 Grafix(그라픽스(Gradient Fixation)의 활용을 설명하며, 이는 스플렉션 컴플렉스에 일시적으로 결합되는 접합 인자 간의 상호 작용을 식별합니다. 이 방법은 시료의 원심분리를 복합체안정화를 위한 고정 시약의 농도증가로 기초한다. 글리세롤 그라데이션에 적재된 효모 총 추출물의 원심분리 후, 회수된 분획은 점블롯에 의해 분석되어 개별 접합 인자의 존재의 현물 및 의결을 위해.

Cwc24-TAP의 퇴적 프로파일을 분석하고 Grafix 방법이 서브콤플렉스를 접합하는 데 결합을 안정화시키는 데 효과적인지 여부를 결정하기 위해, 우리는 글리세롤 그라데이션에 원심분리를 통해 Cwc24-TAP를 표현하는 세포의 총 효모 추출물을 분리하여 교차 연결제로 글루타랄데히드의 존재 또는 부재에 있다. 24μL 분획의 샘플은 TAP 태그의 CBP 부분에 대한 항체를 가진 슬롯 블롯에 의해 분석되었다. 결과?...

Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

여기서는 크로스링커가 있는 글리세롤 그라데이션 원심분리인 그라픽스(Gradient Fixation)의 활용을 설명하여 쌍향적인 복합체에 일시적으로 결합하는 접합 요인 간의 상호 작용을 식별합니다.

사카로미세스 세레비시아스 스플리케소터 서브콤플렉스의 과도성 행위자 검출을 위한 그라픽스의 활용

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

utilization-grafix-for-detection-transient-interactors-saccharomyces

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

3.5K 조회수.  University of Sao Paulo. 여기서는 크로스링커가 있는 글리세롤 그라데이션 원심분리인 그라픽스(Gradient Fixation)의 활용을 설명하여 쌍향적인 복합체에 일시적으로 결합하는 접합 요인 간의 상호 작용을 식별합니다.

비디오: Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

장기 이식에서 HLA Alloantibodies의 검출에 대 한 펩 티 드 배열 맞춤

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

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Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

공부 하는 단백질 키 니 아 제 RNA 포유류 세포 주기 동안 활성의 RNA 인터

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

차에서 파생된 새로 설립된 세포 현탁액 배양에서 6개의 전신 살충제의 물질대사에 관한 연구(동백나무시넨시스 L.) 잎

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

로 키나아제 활성의 일시적인 억제를 통해 인간 유도 만능 줄기 세포 유래 심근 세포의 생착을 강화

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

탠덤 질량 분석과 결합 된 액체 크로마토그래피를 사용하여 사카로 미세화세세비시아의 세포 지질의 정량분석

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

식물 MicroRNAs의 검출 및 정량화를 위한 RNA 블롯 분석

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

베이커효모사카로미세스 세레비시아에 미토콘드리아 게놈 발현 제품의 라벨링 및 시각화

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

탠덤 질량 분광과 결합 된 액체 크로마토그래피를 사용하여 사카로미세스 Cerevisiae의 정량적 Metabolomics

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

대사 라벨링 및 얇은 층 크로마토그래피에 의한 사카로미세스 세레시아의 중립 지질 합성 분석

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.