1. DNA의 Quantitation 절차 <ol><li> 악기를 켭니다. </li><li> PC의 바탕 화면에서 BioSpec - 나노 소프트웨어를 여십시오. </li><li> 간단한 핵산 - dsDNA 탭 (그림 1)를 클릭하십시오. </li><li> 소프트웨어 악기와 통신을 설정하고 초기화 순서를 실행합니다. 이것은 악기 사양에 따라 수행되었는지 확인합니다. </li><li> pathlength 슬라이더에서 0.7 또는 0.2 mm를 선택합니다. 이것은 pathlength를 선택하고 모든 계산이 선택 pathlength를 기반으로합니다. </li><li> BioSpec - 나노 소프트웨어 설치를 클릭 - Autowiping 설정에서 다음. 1 또는 2를 선택합니다. </li><li> 피펫 2 μL (0.7 mm)을 H 2 O 또는 받침대에 버퍼. </li><li> 빈 측정 공백 또는 F6을 누릅니다. 새로운 pathlength 때마다 그것이 공백을 녹화하는 데 필요한 선택됩니다. </li><li> 받침대에있는 샘플의 피펫 2 μL. </li><li> 소프트웨어 시작하거나 악기의 시작 버튼을 누르면 클릭하십시오. 어퍼 창이 샘플 비말 접촉을 이동합니다. 제논 섬광을 볼 수 있습니다. </li><li> 재현성을 결정하기 위해 동일한 샘플에게 3 번 반복합니다. </li><li> 기록된 모든 데이터는. 버드 파일로 저장됩니다. </li><li> . PDF 또는. csv 파일로 저장하려면 PDF / CSV 또는 F9 저장을 클릭하십시오. 결과는 또한 저장 탭에서 적절한 선택을 사용하여. csv 파일로 저장할 수 있습니다. </li></ol> 2. 단백질 Quantitation을위한 프로토콜 <ol><li> 단백질 Quantitation 탭을 선택합니다 (그림 2). </li><li> 알려진 경우, 멸종 계수 e280을 입력합니다. </li><li> e280이 알려져 있지 않은 경우가 e280Calc를 사용하여 계산합니다. </li><li> pathlength 슬라이더에서 0.7 또는 0.2 mm를 선택합니다. </li><li> 설정 - Autowiping 설정을 클릭합니다. 2 선택하거나 위. </li><li> 피펫 3 μL (0.2 mm)을 H 2 O 또는 받침대에 버퍼. </li><li> 빈 측정 공백 또는 F6을 누릅니다. </li><li> 페데 스탈에서 단백질 샘플의 피펫 3 μL. </li><li> 소프트웨어 시작하거나 악기의 시작 버튼을 누르면 클릭하십시오. </li><li> 재현성을 결정하기 위해 동일한 샘플에게 3 번 반복합니다. </li><li> 기록된 모든 데이터는. 버드 파일로 저장됩니다 </li><li> . PDF 또는. csv 파일로 저장하려면 PDF / CSV 또는 F9 저장을 클릭하십시오. </li></ol> 3. 대표 결과 : 1. DNA의 Quantitation에 대한 검색 결과 그림 3은 전형적인 이중 좌초된 DNA 샘플에 대한 측정에서 데이터를 보여줍니다. 농도와 계산 OD 280분의 260 비율도 표시됩니다. 그림 4는 DNA의 quantitation에 대한 재현성 테스트 결과가 포함되어 있습니다. 그림 3 및 4 보여주는 결과의 데이터는 0.26 %의 낮은 상대 표준 편차에서 분명 같은 높은 재현할 수 있습니다. 주 1 : 의 DNA quantitation의 부정확은 자외선 영역에서 흡수 수 있습니다 버퍼 구성 요소의 존재에서 발생할 수 있습니다. 그것은 또한 동질적인 샘플 솔루션을 사용해야합니다. pipetted되는 샘플의 양이 어떤 거품의 도입없이 정확한 볼륨만이 1-2 μL, 조심 pipetting이므로 필요합니다. 2. 단백질 Quantitation에 대한 검색 결과 그림 5는 BSA에 대한 단백질 농도 분석을 나타냅니다. μg / ML 단백질 농도는 e280 가치에 기반을두고 있습니다. 그림 6은 재현성 분석을 보여줍니다. DNA뿐만 아니라 단백질을 위해뿐만 아니라 - 그것은 BioSpec - 나노 정확하게 농도를 측정하는 것을 0.51 %의 상대 표준 편차에서 분명하다. 2 주 : 단백질 농도에 영향을 미치는 요인 단백질의 정확한 농도 결정은 산도, 버퍼, 온도, 또는 첨가제에 의한 불안정성에서 최소한의 효과가있을 경우 가능합니다. 대부분의 단백질 정화 처리의 다양한 단계를 통해 이뤄지는, 따라서 그것은 단백질 비말 형성에 방해가 될 수있는 계면 활성제 또는 다른 첨가제를 포함할 수도 있습니다. 이러한 샘플은 5mm의 pathlength의 석영 세포를 사용하면 매우 좋습니다. <img src="/files/ftp_upload/2699/2699fig1.jpg" alt="그림 1" /> 더블 좌초의 DNA quantitation를위한 그림 1. Analyte 선택 창 <img src="/files/ftp_upload/2699/2699fig2.jpg" alt="그림 2" /> 비 표시 단백질 quantitation에 대한 그림 2. Analyte 선택 창 <img src="/files/ftp_upload/2699/2699fig3.jpg" alt="그림 3" /> DNA의 quantitation를위한 그림 3. 대표 데이터 <img src="/files/ftp_upload/2699/2699fig4.jpg" alt="그림 4" /> DNA의 quantitation를위한 그림 4. 재현성 데이터 <img src="/files/ftp_upload/2699/2699fig5.jpg" alt="그림 5" /> 그림 5. 단백질 quantitation를위한 대표 데이터 <img src="/files/ftp_upload/2699/2699fig6.jpg"고도는 = &quot;그림 6&quot;/&gt; 그림 6. 단백질 quantitation에 대한 재현성 데이터

<ol>
	<li>Aisubel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Short Protocols in Molecular Biology. , (1999).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+and+predict+molar+absorption+coefficient+of+a+protein.">How to measure and predict molar absorption coefficient of a protein.</a> Protein Science. 4, 2411-2423 .</li></ol>

Suja Sukumaran이 박사는이 문서에 등장하는 악기를 만드는 Shimadzu의 직원입니다.

BioSpec - 나노는 등 일관성없는 결과와 지루한 수동 노동의 DNA quantitation 방법 고유의 현재의 문제를 해결하기 위해 설계되었습니다. 각 측정 경험 분자 생물학이 분 3-6에 대한 여러 샘플을 분석할 수 있도록 불과 3 초 정도 걸립니다. 독특한 와이퍼 기능 측정의 처리량을 향상뿐만 아니라, 또한 측정 사이의 무시할 수 carryover의 결과로, 받침대의 수동 청소에 대한 필요성을 제거합니다. 또한, 1000 NG / μL를 포함하는 DNA 샘플에 대한 단 2 잎사귀를 사용하여 테스트, carryover을 표명 미만 2 μL / NG. 5mm의 pathlength 세포의 가용성 희석 핵산과 단백질 샘플을 분석하는 옵션을 제공하며 다른 응용 프로그램이 악기의 사용을 확장합니다.

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concentration determination of nucleic acids and proteins using the micro-volume bio-spec nano spectrophotometer

핵산 quantitation 절차는 지난 30 년간 크게 고급 있습니다. 점점 더 많은, 분자 생물학 자들이 실험에 대한 핵산 샘플 일관성있는 작은 볼륨 분석을 필요로합니다. BioSpec - 나노는 전문 광학과 민감한 PDA 검출기를 통해 현재의 DNA quantitation 방법 고유의 부정확, 비 재현할 결과의 문제에 잠재적인 솔루션을 제공합니다. BioSpec - 나노도함으로써 광섬유 요소 및 수동 청소, 지루한 반복하고, 일관성없는 배치를 제거, 설치, 측정, 및 세척이 장비에 의해 수행되는 이러한 자동화된 기능이 있습니다. 본 연구에서는, 데이터는 DNA와 단백질뿐만 아니라 측정 재현성 및 정확도에의 부량에 제공됩니다. 자동 샘플 연락처 및 빠른 스캔 뛰어난 처리량의 결과, 3 초 안에 측정이 가능합니다. 데이터 분석 소프트웨어의 내장 기능을 사용 수행됩니다. DNA 농도를 계산에 사용되는 공식은 다음과 같습니다 : 샘플 농도 = DF ° (OD260 - OD320) · NACF (1) = 샘플 희석 계수와 NACF = 핵산 농도 계수 어디 DF. 핵산 농도 계수는 1을 선택 analyte에 따라 설정됩니다. 단백질 농도 결과는 μg / ML 또는 각각 e280 및 분자량 값을 입력하여 몰스 / L로 표현할 수있다. 티르, TRP 그리고 시스테인 (SS 결합)에 대한 잔류 값이 e280Calc 탭에 입력하는 경우, 흡광 계수 값은 e280으로 계산됩니다 = 5,500 X (TRP의 잔류물) + 1490 X (티르의 잔류물) + 125 X (시스테인 SS 결합) . e280 값은 농도 계산을위한 소프트웨어에 의해 사용됩니다. 핵산과 단백질의 농도 결정뿐만 아니라, BioSpec - 나노는 다른 많은 analytes에 대한 매우 마이크로 볼륨 분광 광도계 또는 5mm의 pathlength 세포를 사용하여 표준 분광 광도계로 사용할 수 있습니다.

Watch this Scientific Journal Video about Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer at JoVE.com

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

의사 소통이 dsDNA와 단백질 quantitation에 대한 BioSpec - UV - VIS 나노 분광 광도계의 정확성과 재현성에 대한 데이터를 제공합니다. 자동 닦는 함수가 처리량과 더 정확한 결과를 위해 최소한의 carryover 결과를 개선하는 동안에도 초소형 볼륨 (1-2 패), 재현성이 우수합니다.

마이크로 볼륨 바이오 사양 나노 분광 광도계를 사용하여 핵산 및 단백질 농도 결정

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

concentration-determination-nucleic-acids-proteins-using-micro-volume

Research

JoVE Journal

Biology

38.8K 조회수.  Scientific Instruments. 의사 소통이 dsDNA와 단백질 quantitation에 대한 BioSpec - UV - VIS 나노 분광 광도계의 정확성과 재현성에 대한 데이터를 제공합니다. 자동 닦는 함수가 처리량과 더 정확한 결과를 위해 최소한의 carryover 결과를 개선하는 동안에도 초소형 볼륨 (1-2 패), 재현성이 우수합니다.

비디오: Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

NanoDrop 2000c 분광을 사용 Microvolume 단백질 농도 결정

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

연구

생물학

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidic Mesophases를 사용하여 구조 결정을위한 막 단백질 Crystallizing

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

동력학 및 구조 기능 분석의 선호도 결정에 대한 올바른 단백질 농도의 중요성

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

히타치 F - 7000 형광 분광 및 PicoGreen 염료를 사용하여 dsDNA의 부량

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

핵산의 NanoDrop Microvolume의 Quantitation

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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 ...

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 ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

를 사용하여 모델 단백질로 비정규 아미노산의 잔류 특정 법인 설립<em&gt; 대장균</em&gt; 휴대없는 전사 번역 시스템

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

야생형과 간암의 시알 산의 결정<em&gt; CMAH</em&gt; 넉 아웃 마우스.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...