미끄러운 바위 대학에서 제공하는 자금. 우리는 그들의 통찰력 T 보론 III (미끄러운 바위 대학)와 C하니 (펜실베니아 대학)에 감사드립니다.

니켈 쉬프-자료 단지 1. 가수 분해 <ol><li> 실온에서 250 mL의 둥근 바닥 플라스크를 교반하면서 N, N- 디메틸 포름 아미드 (DMF) 40 mL를 상기의 Ni-PBP - 쉬프 염기 착체 1 밀리몰 녹인다. </li><li> 수성 0.2 M EDTA 용액의 pH 4.5의 60 mL를 넣고. </li><li> 자기 교반 막대를 이용하여 플레이트를 교반 밤새 합성 용액을 교반한다. 쉬프 염기 복합 가수 분해로, 색상은 흰색에 깊은 적색에서 이동합니다. </li><li> 모든 적색 착색의 부재에 의해 나타낸 바와 같이, 반응 종결 후, 250 mL의 분액 깔때기에 반응 옮긴다. </li><li> 50 mL의 디클로로 메탄을 추가 분액 깔때기 캡 섞는다. 폐기물 비이커에 유기 세정 배수. PBP 및 잔여 니켈 PBP-쉬프 염기 단지를 제거하려면이 과정을 세 번 반복합니다. 250 mL의 둥근 바닥 플라스크에 남아있는 수성 층을 수집. </li></ol> 가수 분해 아미노산의 2 FMOC 보호 <ol><li&gt; 고체 중탄산 나트륨을 사용하여 pH 7로 상술 한 단계 150에서 분리 된 수성층을 조정한다. pH가 정기적으로 산도 테스트 스트립을 사용하여 확인합니다. </li><li> 용액에 168 mg의 탄산 수소 나트륨 (2.00 밀리몰, 2 당량)을 첨가하고 자기 교반 막대를 이용하여 교반하고 플레이트를 교반한다. </li><li> 10 ML 비얼에서 디 옥산 최소량 (약 4-5 ㎖)에 337 mg의 N의 FMOC -hydroxysuccinimide 에스터 (1.00 밀리몰, 1 당량)을 녹인다. 수용액이 용액을 전송하고, 밤새 교반 하였다. </li><li> 반응물을 밤새 교반시킨 후, 1 M 염산으로 pH 2로 생성 된 용액을 산성화. pH가 정기적으로 산도 테스트 스트립을 사용하여 확인합니다. </li><li> 250 ㎖의 분액 깔때기에 옮기고, 반응을 50 ㎖의 에틸 아세테이트를 추가한다. 상기 분별 깔때기 캡 잘 혼합하고, 250 mL의 삼각 플라스크에 유기층을 수집한다. 유기 추출물을 결합,이 과정을 두 번 더 추가를 반복합니다. 마그네슘 sulfat 대략 3g 합한 유기 추출물을 건조이자형. </li><li> 조 FMOC 보호 된 아미노산을 수득하는 회전 증발기를 사용하여 합한 유기 추출물을 농축시킨다. </li></ol>

<ol>
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A., Kelly, D. E., Wolfe, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+syntheses+of+Fmoc-protected+non-proteogenic+amino+acids:+useful+building+blocks+for+combinatorial+libraries.">Large-scale syntheses of Fmoc-protected non-proteogenic amino acids: useful building blocks for combinatorial libraries.</a> Org. Process Res. Dev. 5 (4), 445-449 (2001).</li><li>Cruz, L. J., Beteta, N. G., Ewenson, A., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&amp;#34;One-pot&amp;#34;,+preparation+of+N-carbamate+protected+amino+acids+via+the+azide.">&amp;#34;One-pot&amp;#34;, preparation of N-carbamate protected amino acids via the azide.</a> Org Process Res. Dev. 8 (6), 920-924 (2004).</li><li>Hart, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ullmann's Encyclopedia of Industrial Chemistry. , (2000).</li><li>Adamson, J. G., Blaskovich, M. A., Groenevelt, H., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+convenient+synthesis+of+tert-butyl+ethers+of+Fmoc-serine,+Fmoc-threonine,+and+Fmoc-tyrosine.">Simple and convenient synthesis of tert-butyl ethers of Fmoc-serine, Fmoc-threonine, and Fmoc-tyrosine.</a> J. Org. Chem. 56 (10), 3447-3449 (1991).</li><li>Seyfried, M. S., Lauber, B. S., Luedtke, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-turnover+isotopic+labeling+of+Fmoc-+and+Boc-protected+amino+acids+with+oxygen+isotopes.">Multiple-turnover isotopic labeling of Fmoc- and Boc-protected amino acids with oxygen isotopes.</a> Org. Lett. 12 (1), 104-106 (2010).</li><li>Bonke, G., Vedel, L., Witt, M., Jaroszewski, J. W., Olsen, C. A., Franzyk, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimeric+building+blocks+for+solid-phase+synthesis+of+&#945;,-peptide-&#946;,-peptoid+chimeras.">Dimeric building blocks for solid-phase synthesis of &#945;,-peptide-&#946;,-peptoid chimeras.</a> Synthesis. 2008 (15), 2381-2390 (2008).</li></ol>

저자가 공개하는 게 없다.

상기 프로토콜은 두 가지 중요한 단계를 중성 pH 조건이 절연 아미노산 후속 FMOC 보호 하에서 니켈 쉬프 염기 착체의 아미노산 골격의 분리를 촉진하는 능력에 유용하다. 첫 번째 단계는 복잡에서 아미노산의 방출을 용이하게하기 위해 EDTA를 함유하는 DMF / 물 용액을 교반 포함한다. 잔류 착체 또는 유기 부산물 쉽게 추출하여 제거 될 수있다. 이 프로토콜의 두 번째 단계에서는 첫 번째 단계에서 ?...

자연에서 발견 스물 개 자연적으로 발생하는 아미노산과 다를 자연스러운 아미노산 (UAA의) 베어링 측쇄 응용 프로그램의 넓은 범위에서 유틸리티를 발견했다. 이러한 UAA 년대의 합성 단, 측쇄의 구조 및 아미노산 주쇄의 입체 화학에 따라 어려울 수있다. 니켈 쉬프 염기 착체의 맥락에서 글리신의 크 본드 활성은 α, β 디아 미노 산 1, 2 또는 헤테로 측쇄 플루오르 UAA의 베어링을 포함한 아미노산 유도체의 다양성을 생성하기 위해 사용되었다. 삼 비 천연 측쇄를 첨가 한 후, UAA의 전형적 염산 (4)에 환류하여 쉬프 염기 착체로부터 제거하고,이어서 이온 교환 크로마토 그래피를 사용하여 분리되어 기능화. 일반적으로 효율적이지만,이 프로토콜은 발생고상 펩티드 합성 (SPPS)에서 사용되기에 부적합 할 수있다 미노 산. SPPS의 자연 그대로 이들 보호기의 UAA와의 분리를 방지 산해리 측쇄 보호기의 존재 및 일반적인 니켈 쉬프 염기 분해 조건 강산성 성질을 필요로한다. 우리의 지식으로, 단 하나의 대체 분해 방법이보고되어있다 : 에틸렌 디아민 테트라 아세트산 (EDTA) 및 하이드라진을 사용 승온에서, 그 자체가 몇몇 측쇄는 프탈이 미드로서 보호기에 적합하지 않을 수 5 조건있다. <img alt="그림 1" src="/files/ftp_upload/55677/55677fig1.jpg" /> 도 1 : 니켈 2+, PBP, 글리신 (의 Gly)에서 니켈의 Gly-PBP의 합성. <a href="http://ecsource.jove.com/files/ftp_upload/55677/55677fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> </A&gt; 여기서 우리는, 니켈의 Gly-PBP (도 1) 니켈 쉬프 염기 착체의 가수 분해하는 방법을보고한다. 니켈 2+, 글리신, 및 피리딘 -2- 카르 복실 산 (2- 벤조일 페닐) - 아마이드 (PBP), 6 유래의이 복합체는 UAA의 다양한 합성에 유용한 플랫폼 증명 용이하다되었습니다 액세스 두 단계의 합성 경로를 사용. 이러한 착체의 합성 문헌 (7) - 이미 기재 고 수율이다. 우리의 결과는 6 이하의 UAA 베어링 산해리 측쇄가 보호기와 사용에 적합한 중성 pH 조건 약산성에서 EDTA를 이용하여 가수 분해 조건의 적용을 보여준다. 가수 분해 후, 생성 된 수성 용액을 분리 할 수 있고 FMOC 보호 된 아미노산을 수득 표준 FMOC 보호 상태 (도 2)에 즉시 실시. 그림 2 : 가수 분해 및 Ni-PBP-의 Gly에서 고립 된 아미노산의 FMOC 보호. 반응 조건 : 전. EDTA (12 당량), 4.5의 pH; II. pH 7로 세척하고 아세트산 에틸 조정; III. FMOC - 오스 (1 당량)의 NaHCO3 (2 당량). <a href="http://ecsource.jove.com/files/ftp_upload/55677/55677fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ni-PBP-Gly</td>
			<td></td>
			<td></td>
			<td>Synthesized from published protocol</td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Fisher</td>
			<td>D119-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher</td>
			<td>S311-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Acros</td>
			<td>AC610050040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-OSu</td>
			<td>Chem-Impex</td>
			<td>&#34;00147&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dioxane</td>
			<td>Fisher</td>
			<td>D111-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Acros</td>
			<td>AC610060040</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEOPrep 60ECO Silica Gel</td>
			<td>ZEOChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hexanes</td>
			<td>Fisher</td>
			<td>3200250.650.443</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography Column</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH Test Strips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Evaporator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Separatory Funnel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Round Bottom Flask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Bar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

hydrolysis of a ni-schiff-base complex using conditions suitable for retention of acid-labile protecting groups

비 천연 아미노산은 일반적으로 자연에서 보지 측쇄 작용기를 함유하는 아미노산 점점 합성 펩티드 서열에서 발견된다. 몇몇 비 천연 아미노산의 합성은 종종 니켈 양이온에 의해 안정화 쉬프 염기로 이루어지는 전구체의 사용을 포함한다. 비정상적인 측쇄이 쉬프 염기 착체에서 발견되는 아미노산 골격 상에 설치 될 수있다. 생성 된 비 천연 아미노산은 전형적 강산성 용액으로 환류를 이용하여, 쉬프 염기의 복잡한 분리하여 가수 분해 될 수있다. 비 천연 아미노산은 마이크로파를 이용한 고상 펩티드 합성에 사용되는 이러한 강산성 조건 산해리 필요한 측쇄 보호기를 제거 할 수있다. 본 연구에서, 우리는 효율적인 가수 분해 및 니켈 쉬프 염기 착체로부터 분리 아미노산 후속 FMOC 보호를 제시한다. 본 연구에 제시된 가수 분해 조건은 산해리의 유지에 적합IDE 쇄 보호기 및 비 천연 아미노산의 다양한 기판에 적용 할 수있다.

우리는 가혹한 pH 조건 없이도 쉬프 염기의 수성 가수 분해 효율을 허용 할 수있는 니켈의 Gly-PBP 복합체에서의 Ni 2+의 제거를 가정. EDTA가 저렴하고 잘 연구 킬레이트 제이기 때문에, 10 우리는 니켈 PBP-의 Gly의 용액에 EDTA를 첨가함으로써 복합체의 분해를 촉진 니켈 2+ 이온의 킬레이트 화를 촉진한다고 가정 하였다. <p class="jove_content" fo:...

Watch this Scientific Journal Video about Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups at JoVE.com

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

여기서, 우리는 효율적인 가수 분해 및 니켈 쉬프 염기 착체로부터 분리 아미노산 후속 FMOC 보호를 제시한다. 산해리 측쇄 보호기의 유지가 요구되는 경우 여기 제시된 가수 분해 조건을 사용하기에 적합하다. 이 기술은 천연 아미노산의 다양한 기판에 적용 할 수있다.

보호 그룹 산 - 불안정의 보존에 적합한 니켈 쉬프 - 자료의 가수 분해 복잡한 사용 조건

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

hydrolysis-ni-schiff-base-complex-using-conditions-suitable-for

Research

JoVE Journal

Biochemistry

9.6K 조회수.  Slippery Rock University.  여기서, 우리는 효율적인 가수 분해 및 니켈 쉬프 염기 착체로부터 분리 아미노산 후속 FMOC 보호를 제시한다. 산해리 측쇄 보호기의 유지가 요구되는 경우 여기 제시된 가수 분해 조건을 사용하기에 적합하다. 이 기술은 천연 아미노산의 다양한 기판에 적용 할 수있다. 

비디오: Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Cryomilled 포유 동물 세포에서 단백질 단지 선호도 캡처

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

연구

생화학

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

안정 동위 원소 추적을 사용하여 복잡한 세포 공동체의 정렬 된 부분 집단에서 대사를 측정하는 방법

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

유 중수 에멀젼의 원심 분리에 의해 거 소포 캡슐화 미소 구의 제조

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

이성질체 단백질 복합체 -DNA 상호 작용 연구를위한 수정 된 효모 원 하이브리드 시스템

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

탄수화물 전송 기질 바인딩 단백질 SP0092의 생 화 학적 및 구조적 특성

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

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 ...

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

엄격하게 혐기성 /안산 조건하에서 미생물의 재배를위한 중간 준비

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

다목적 미세 유체 칩의 직렬 결정학에 의한 효소의 결정화 및 구조적 결정: 기질 복합체

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

구슬에 갈렉틴-3 - U1 snRNP 복합체에 의한 접합 활동의 보완

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

박테리아의 RIBO-seq: NGS 시퀀싱을 위한 샘플 수집 및 라이브러리 준비 프로토콜

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...