우리는 이네스 Racké의 기술 지원을 인정하고 싶습니다.

1 부 : CBB의 얼룩 솔루션의 준비 <ol><li> CBB의 60-80 MG는 G - 250은 2-4시간에 대한 감동으로 bidistilled 물 1리터에 녹아 있습니다. 마지막으로, 집중 HCL 3 ML은 다른 분간 교반과 함께 진한 파란색 솔루션에 추가하고 나중에 사용하기 위해 어둠에 저장됩니다. 이 솔루션은 그 얼룩의 효율성을 잃지 않고 몇 개월까지 주 동안 최대 저장할 수 있습니다. </li><li> 집중 HCL은 퓸 후드에서 사용하는 일반적인 치료 깨물어으로 취급해야합니다. 최종 솔루션은 산도 약 2 있으므로 장갑을 사용해야하고 피부와의 접촉을 피해야한다에있을 것입니다. </li></ol> 2 부 : SDS - PAGE <ol><li> 단백질 시료의 적절한 aliquots는 1X 로딩 버퍼의 최종 농도 버퍼를 읽어와 혼합됩니다. 우리는 125mM Tris/H3PO4 (25 ° C에서 산도 7.5), 2mM EDTA (에틸렌 다이아 민 테트라 초산), 4% SDS, 200mM DTT, 0.02 % bromophenol 파란색과 50 %의 글리세롤과 2X 로딩 버퍼를 사용합니다. SDS - PAGE 다른 로딩 버퍼 잘으로 사용할 수 있습니다. </li><li> 단백질 샘플이 로딩되기 전에 약 5 분 가열합니다. 한편, 겔 전기 영동 챔버는 실행을위한 준비가되어 있습니다. 우리는 XCell SureLock에 © 비스 - 트리스 젤 (Invitrogen) ® MES - 버퍼와 미니 셀 (Invitrogen)가 실행 버퍼지만 다른 겔 및 전기 시스템뿐만 아니라 사용할 수있는 프리 캐스트 4-12%의 NuPAGE를 사용합니다. </li><li> 단백질 샘플은 220V 50 분 실행 젤과 전기 영동에로드됩니다. </li></ol> 파트 3 : 젤의 스테 이닝 <ol><li> 겔 카세트가 해체되고 젤은 이후 세정 단계를 상자에 배치됩니다. </li><li> bidistilled 물을 약 100 ML은 겔에 추가하고 30 초 동안 전자 레인지에 가열합니다. 비등가 발생하기 전에 난방을 중단해야합니다. 젤있는 상자는 다음 3-5 분 쉐이크에 위치합니다. 이 세척 단계는 신선한 물로 두 번 반복됩니다. </li><li> 충분 CBB의 얼룩 솔루션은 상자에 겔 10 초위한 전자 레인지에 가열 상자를 커버에 추가됩니다. 끓는없이. 젤있는 상자 후 얼룩을 마무리를위한 쉐이크에 위치합니다. 이미 일분 후, 단백질 밴드는 15-30 분 후 볼 수 있습니다. 얼룩은 대부분의 경우 충분히 강하. </li><li> 얼룩 솔루션은 해제 붓고되고 50-100 ML bidistilled 물을 더 쉐이크에서 젤의 하늘색 배경을 destain하기 위해 추가됩니다. 물이 필요한 경우 추가 destaining 신선한 물로 교체하실 수 있습니다. </li><li> 젤은 스캔한 사진이나 장기 보관을위한 건조 수 있습니다 </li></ol> 4 부 : 대표 결과 : 그림을 참조하십시오. 설명 절차에 따라 제대로 스테인드 젤 1. 그림을 참조하십시오. 얼룩 및 잔류 SDS하기 전에 충분히 세탁되지 않은 젤에 대한 2 효율적으로 얼룩을 억제. 마커 차선은 (*) 마커 단백질 같은 금액을 포함합니다. <img src="/files/ftp_upload/1350/Fig1.png" alt="" /> 그림. 1 : 단백질 정제의 샘플 (분자량 마커 *)를 로딩 후 대표 겔 얼룩. <img src="/files/ftp_upload/1350/Fig2.png" alt="" /> 그림. 2 : CBB의 얼룩하기 전에 충분히 세탁되지 않은 CBB 스테인드 젤. 단백질 밴드가 (마커 레인 *이 그림에서와 같이 단백질의 같은 양의 1. 포함되어 있습니다) 약한 나타납니다.

<ol>
	<li>Wondrak, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+for+fast+visualization+of+protein.">Process for fast visualization of protein.</a> US patent. , (2001).</li><li>Wondrak, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+for+fast+visualization+of+protein.">Solution for fast visualization of protein.</a> US patent. , (2001).</li></ol>

아무 충돌 관심 없습니다. 절차 (참조을 참조하십시오.) 원래 EM Wondrak하여 특허 출원에 출판 되었음 : 위에서 설명한.

<ul><li> 세척 단계는 단백질의 효율적인 얼룩을 위해 중요하다. 2 분 또는 물 (&lt;50ml)의 감소 볼륨 아래의 감소 세척 시간은 겔에 잔류 SDS 높은 금액으로 인한 가능성이 가장 높은 하늘색 단백질 밴드에 발생할 수 있습니다. </li><li> 단백질은 질량 분광법으로 분석 될 경우, 전자 레인지에서 가열 단계는 생략한다, 10의 각 단계에서 분 및 밴드 강도까지 연장 얼룩 시간까지 확장 젤의 세탁 시간은 충분히 강하 . 질량 분?...

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<td>Coomassie Brilliant Blue G-250</td>
<td>&nbsp;</td>
<td>AppliChem</td>
<td>A3480</td>
<td>any other CBB G-250 could be used as well</td>
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<td>Concentrated HCl</td>
<td>&nbsp;</td>
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staining of proteins in gels with coomassie g-250 without organic solvent and acetic acid

쿠매시 블리언트 블루 (CBB), 독성 및 인화성 유기 용제 (메탄올, 에탄올 또는 2 - 프로파놀)과 초산 높은 내용으로 솔루션을 사용하는 고전적인 단백질 얼룩 프로토콜에 얼룩 및 SDS 후 젤의 단백질 destaining, 고정에 사용됩니다 페이지. 짧은 시간 동안 전자렌지에 얼룩 솔루션을 가열, 절차를 속도를하는 것은 자주 사용됩니다. 독성 또는 유해 메탄올, 에탄올 또는 2 - 프로파놀 안전 고려 사항으로 인해 피해야한다 실험실에서 초산의 강한 냄새의 증발이 일반적으로 발생합니다. 원래 EM Wondrak (US2001046709 (A1), US6319720 (B1))하여 두 특허 출원에 발표된 프로토콜에서 얼룩 솔루션의 대체 성분이 더 용매 유기 또는 산성이 사용되지하는에 설명되어 있습니다. CBB는 bidistilled 물속에 녹아있는 것입니다 (CBB G - 250 당 리터의 60 80mg) 35 MM HCL은 얼룩 솔루션에서 유일하게 다른 화합물로 추가됩니다. 젤의 CBB의 staning는 bidistilled 물에 젤의 SDS - PAGE와 철저한 세척 후에 이루어집니다. 세탁 중에 젤 가열 및 단계를 얼룩으로, 프로세스는 빨리 완료 될 수 있으며, 아무런 독성 또는 유해 compunds이 증발되지 않습니다. 단백질 얼룩이 솔루션을 얼룩에 젤 가열 후 일분 이내에 이미 발생하고 완전히 스테인드 단백질에 영향을주지 않고, bidistilled 물속에 스테인드 젤의 장기간 세척하여 완전히 destained하는 약간 파란색 배경과 15-30 분 후에 개발 밴드.

Watch this Scientific Journal Video about Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid at JoVE.com

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

쿠매시 블리언트 블루 (CBB)의 polyacrylamide 젤의 G - 250로 단백질 얼룩에 대한 짧은 프로토콜은 CBB와 고전적인 얼룩 절차에서와 같이 유기 용제 또는 초산을 사용하지 않고도 설명되어 있습니다.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

staining-proteins-gels-with-coomassie-g-250-without-organic-solvent

Research

JoVE Journal

Biology

52.8K 조회수.  EMBL Heidelberg. 쿠매시 블리언트 블루 (CBB)의 polyacrylamide 젤의 G - 250로 단백질 얼룩에 대한 짧은 프로토콜은 CBB와 고전적인 얼룩 절차에서와 같이 유기 용제 또는 초산을 사용하지 않고도 설명되어 있습니다.

비디오: Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

젤의 단백질 염색법

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

연구

생물학

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Polyacrylamide의 젤의 단백질에 대한 신속하고 민감한 콜로이드 쿠매시 G - 250 스테 이닝

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Methyltransferases의 프로 파일링 및 기타 S - adenosyl - L</sub캡처 컴파 운드 질량 분광법 (CCMS)에서> - 호모 시스테인 결합 단백질

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

막 단백질의 Conformational 역학 검사 현장에서 사이트 이동 형광 라벨링과

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

환경 샘플에서 유기 P의 높은 처리량 측정 및 분류

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

펩타이드 면역 친 화성 농축을 사용하여 단백질의 양을 정함은 질량 분광법과 함께

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 Their redox chemistry is even younger, just about ten years old, but despite this short history these deltahedral clusters ions E9n- (E = Si, Ge, Sn, Pb; n = 2, 3, 4) have already shown interesting and diverse reactivity and have been at the forefront of rapidly developing and exciting new chemistry.3-6 Notable milestones are the oxidative coupling of Ge94- clusters to oligomers and infinite chains,7-19 their metallation,14-16,20-25 capping by transition-metal organometallic fragments,26-34 insertion of a transition-metal atom at the center of the cluster which is sometimes combined with capping and oligomerization,35-47 addition of main-group organometallic fragments as exo-bonded substituents,48-50 and functionalization with various organic residues by reactions with organic halides and alkynes.51-58 
This latter development of attaching organic fragments directly to the clusters has opened up a new field, namely organo-Zintl chemistry, that is potentially fertile for further synthetic explorations, and it is the step-by-step procedure for the synthesis of germanium-divinyl clusters described herein. The initial steps outline the synthesis of an intermetallic precursor of K4Ge9 from which the Ge94- clusters are extracted later in solution. This involves fused-silica glass blowing, arc-welding of niobium containers, and handling of highly air-sensitive materials in a glove box. The air-sensitive K4Ge9 is then dissolved in ethylenediamine in the box and then alkenylated by a reaction with Me3SiC&equiv;CSiMe3. The reaction is followed by electrospray mass spectrometry while the resulting solution is used for obtaining single crystals containing the functionalized clusters [H2C=CH-Ge9-CH=CH2]2-. For this purpose the solution is centrifuged, filtered, and carefully layered with a toluene solution of 18-crown-6. Left undisturbed for a few days, the so-layered solutions produced orange crystalline blocks of [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en which were characterized by single-crystal X-ray diffraction. 
The process highlights standard reaction techniques, work-up, and analysis towards functionalized deltahedral Zintl clusters. It is hoped that it will help towards further development and understanding of these compounds in the community at large.

We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.

유기 그룹과 아홉살 원자 Deltahedral Zintl 게르마늄의 이온과 작용화의 합성

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

3DNA과 핵산의 구조를 분석하고 구축

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Zebrafish의 유충 루시 페라 제 리포터 시스템과 글루코 코르티코이드 신호에 대한 화학적 스크리닝 절차

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...