支持这项研究是由美国能源署（DE - FG02 - 09ER09 - 08和DE - PS02 - 09ER09 - 18），在加州大学洛杉矶分校琼森综合癌症中心，工业大学合作研究计划“（市政局发现批准，bio07 -10665）。我们感谢在加州大学洛杉矶分校生物医学回旋加速器设施Nagichettiar Satyamurthy博士和工作人员提供的F - 18的放射性同位素和许多有见地的讨论。我们感谢博士。迈克尔柯林斯，格雷格 - 勒布朗，约瑟夫兰伯特，和CEM的Barnhardt凯勒从他们的技术咨询和支持。我们感谢达林德克 - 威廉姆斯，威廉姆斯，博士。香港敦林，约瑟夫迈克尔面包车大坝的设计和加工零件修改CEM微波反应器，用于固相萃取净化模块。

1。前期准备<ol><li> V -小瓶（5毫升）RV1（搅拌棒）是用来作为主要执行微波合成反应釜。它是连接到PEEK的适配器七进/出端口连接，放在里面的微波腔（见图2）。 RV2连接到SPE柱（一）收集的原油[18楼 ] SFB的。 RV3连接到SPE柱（二）收集的最后18架F] SFB的解决方案。它可以被放置在一个温水浴（40℃）前集中相应的解决方案，在PBS缓冲液中重组，尤其是对下游的放射性标记的生物分子。 </li><li> 收集原油[18 F] SFB：填充乙腈/ H 2 O [6毫升; 1:4（V：V）]的解决方案，5％的水醋酸（8毫升），乙腈水库一（2毫升），设置B和C分别。然后激活一个SPE柱（一）（聚苯乙烯，默克LiCholut EN）与乙醇（10毫升），5％水溶液的醋酸（10毫升）洗。 </li><li> 设置收集纯化[18楼 ] SFB：准备水库D和E与H 2 O和10毫升3毫升乙醚填写。第二个SPE柱（二）（聚苯乙烯，默克LiCholut EN）被激活上述相同的步骤。 </li><li>启动预调节的高效液相色谱仪（洗脱缓冲液：乙腈/ H 2 O，1:1（V / V）含0.2％TFA的;流速：3毫升/分）的高效液相色谱法，色谱柱[反相半预习列（LUNA，5微米C18（2）100 nm，250 × 10毫米），Phenomenex，托伦斯，美国加利福尼亚州。 </li></ol> 2。干涸[即非运营商补充说，（NCA）] [18 F]氟的制备<ol><li> [18楼 ] [18 O] H 2 O（100μL），氟化钠溶液添加到Kryptofix 222（20毫克），1M水溶液K 2 CO 3（26μL）和乙腈（0.8毫升）的混合物在一个Eppendorf公司管。整个解决方案，然后充分混合，然后转移到通过入口1号线RV1。 [18楼 ]氟化钠溶液也可通过阴离子交换盒（如QMA光九月乐从水中）陷阱氟- 18，然后在乙腈的混合物的K 2 CO 3和Kryptofix洗脱。 </li><li>下微波控制程序执行的干燥序列（20W，3分钟），以消除RV1 [真空条件下残留的水。后冷却系统的温度低于50℃，额外的乙腈（1.0毫升）引入反应堆和序列是重复一次。 </li></ol> 3。合成乙基4 - [18楼 ] fluorobenzoate <ol><li>一个DMSO溶液（0.4毫升）含乙基4 - （N，N，N -三甲基）三氟苯甲酸（1.5毫克）加入RV1通过进线2。 </li><li>下搅拌，容器冷却和所有阀门的微波控制程序执行的标签序列（50W，1分钟）封闭负担乙基4 - [18 F] fluorobenzoate（[18 F]，2） 。 </li></ol> 4。合成的钾4 - [18楼 ] fluorobenzoate <ol><li>一个DMSO溶液（0.5毫升）含KOtBu（13毫克）加入RV1通过进线3。 </li><li>下搅拌，容器冷却和所有阀门的微波控制程序执行Deprotect方案（40瓦，1分钟）封闭负担4 - [18楼] fluorobenzoate盐（[18楼] 3） 。 </li></ol> 5。合成原油[18 F] SFB <ol><li>乙腈溶液（2.5毫升）含TSTU（30毫克）被添加到RV1通过进线6。 TSTU是水分和光敏感。应该分装到小瓶中，并在4 ° C铝箔盖的密闭容器中储存。 </li><li>下搅拌，容器冷却和所有阀门的微波控制程序执行封闭耦合序列（30W，2分钟）[18楼] SFB的负担粗。 </li></ol> 6。 SPE纯化[18楼 ] SFB的制备<ol><li> 5％的水醋酸（1.0毫升）被添加到RV1通过进口第7行中反应混合物。解决的办法是再转移到小瓶含8毫升5％的醋酸水溶液（图2）乙。 </li><li>通过SPE柱（一）稀释反应混合物陷阱原油[18楼 ] SFB的使用氮气（10磅）。 </li><li>洗SPE柱用乙腈和H 2 O [10毫升，1:4（V：V）]的混合物水库答：（一） </li><li> [18楼 ] SFB的洗脱RV2从水库C.使用乙腈（2毫升） </li></ol> 7。原油净化[18 F] SFB的电台高效液相色谱法<ol><li>稀要么原油[18 F] SFB或SPE纯化[18 F] SFB在RV2与H 2 O（2毫升），并转移到HPLC循环（5毫升）的混合物。的解决方案是无线电HPLC乙腈/ H 2 O，1:1（V / V）含0.2％TFA的流速注入3毫升/分钟]。 </li><li>收集含有纯化[18 F] SFB（保留添分数E：8-10分钟）到小瓶D（与10毫升的H 2 O预填充）（图2）。关键的一步：如果正确执行，这里收集的比例量应该是4-5毫升。 </li><li>通过SPE柱（二）稀释反应混合物陷阱纯化[18楼 ] SFB的使用氮气（10磅）。干燥的氮气流2-3分钟的墨盒。 </li><li> [18楼 ] SFB洗脱RV3从水库E.乙醚（3毫升）。 </li><li>溶剂蒸发RV3干燥的氮气温柔流使用水浴（40℃）（10磅）。最后干[18楼 ] SFB可重组PBS缓冲液的下游应用。 </li></ol> 8。代表性的成果： 我们开发了一种简化，快速，一锅煮合成方法[18 F] SFB的使用在每放/化学转化一个无水条件下和微波加热脱保护策略。图1显示了我们的放射合成的细节。最终产品的身份证实了高效液相色谱保留时间比较，与非放射性的SFB的参考。纯化[18楼 ] SFB还通过广播，薄层色谱法和高效液相色谱法分析，以确定其放射化学和化学纯度。 RCY [18 F] SFB时后60分钟内35 ± 5％HPLC纯化（N&gt; 30），放射化学纯度高（&gt; 99％）和良好的化学纯度（HPLC的配置文件中看到紫外线跟踪，图3 ）。 CA的具体活动。 67-330吉贝/μmol（1.8-9.0次/μmol），取决于起始放射性。 <img src="/files/ftp_upload/2755/2755fig1.jpg" alt="图1" /> 图1微波辅助一锅煮放射合成[18 F] SFB。首先，radiofluorination乙基4 - （N，N，N -三甲基）三氟苯甲酸（1）下进行微波加热（50瓦，1分钟）的存在[K⊃2.2.2] [18楼 ]楼-在二甲基亚砜（DMSO）的复杂负担乙酯4 - [18 F] fluorobenzoate（[18 F]，2） 。没有净化，叔丁醇钾（T BuOK）的DMSO溶液添加和反应容器微波照射（40瓦，1分钟）完成无水脱保护。最终转换[18 F]分为3 [18 F] SFB时使用- O -（N -琥珀酰亚胺） - N，N&#39;，N&#39; - tetramethyluronium四氟（TSTU）激活。在乙腈TSTU被添加到反应混合物中，含有4 - [18楼] fluorobenzoate（[18 F]，3）盐，这最后的合成一步取得原油 [18楼] SFB的加热后（30瓦，2分钟）。 <img src="/files/ftp_upload/2755/2755fig2.jpg" alt="图2" /> 图2设置微波辅助一锅煮[18楼 ] SFB的合成示意图。 <img src="/files/ftp_upload/2755/2755fig3.jpg" alt="图3" /> 图3：最后的无线电HPL色谱[18 F] SFB 。顶：在254 nm的紫外信号;底部：放射性信号;插图：紫外线在254 nm（X 33.3）的信号。 <img src="/files/ftp_upload/2755/2755table1.jpg" alt="表1" /> 表1总结[18 F] SFB radiosyntheses在文献报道使用烷基4 - （三甲基）苯甲酸作为前体三氟。

这种方法已提交美国专利申请。

这种简化的三步，一锅煮的18架F -酰化试剂放射合成[18 F] SFB的是开发基于非水溶液化学。这个过程具有优异的可重复性和可用于生产可靠[18 F] SFB的自动化放射化学模块，由于两个关键的修改，如以下所述：1 。我们聘请在无水KOtBu / DMSO系统去保护/皂化的步骤，以取代普通的水基本或酸性溶液。我们的非水性脱保护策略，使固相萃取净化，没有中间或溶剂蒸发/交换顺序加?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>在水溶液中的乙酸（5％，V / V） </td><td>费舍尔 </td><td> A38 - 500 </td><td>我们的实验室编制</td></tr><tr><td>乙腈</td><td> Sigma - Aldrich公司</td><td> 75-05-8 </td><td></td></tr><tr><td>乙醚</td><td> Sigma - Aldrich公司</td><td> 14775 </td><td></td></tr><tr><td>二甲基亚砜（DMSO） </td><td> Sigma - Aldrich公司</td><td> 472301 </td><td></td></tr><tr><td>乙基4 - （N，N，N -三甲基）苯甲酸三氟</td><td>编制在实验室</td><td></td><td></td></tr><tr><td> 4,7,13,16,21,24 - Hexaoxa -1,10 - 二氮杂双环[8.8.8]二十六烷（K222） </td><td> Sigma - Aldrich公司</td><td> 29,111-0 </td><td></td></tr><tr><td> O - （N -琥珀酰亚胺） - N，N，N&#39;，N&#39; - tetramethyluronium四氟（TSTU） </td><td> Sigma - Aldrich公司</td><td> 105832-38-0 </td><td></td></tr><tr><td>碳酸钾水溶液（1M） </td><td> Sigma - Aldrich公司</td><td> 209619 </td><td>我们的实验室编制</td></tr><tr><td>钾叔丁醇</td><td> Sigma - Aldrich公司</td><td> 156671 </td><td></td></tr></tbody></table>

microwave-assisted one-pot synthesis of n-succinimidyl-4-[18f]fluorobenzoate ([18f]sfb)

生物分子，包括肽，蛋白质1-9，10,11和抗体及其设计片段，12-14获得作为潜在的治疗和分子显像剂的重要性。值得注意的是，与正电子发射放射性同位素标记（例如，铜64，GA - 68或F - 18）时，他们可以被用来作为许多生理和病理过程中有针对性的成像探测。15-18因此，重大努力用于18架F -标记的生物分子的合成和探索。虽然也有直接的 18架F -标签肽的优雅的例子，19日至22日在苛刻的反应条件（即，通常是不相容的有机溶剂，极端的pH值，高温）与直接radiofluorination相关的脆弱的蛋白质样品。因此，到目前为止，纳入到生物分子的放射性标记的假肢群体仍然是首选的方法 。23,24 N -琥珀酰亚胺- 4 - [18 F] fluorobenzoate（[18楼 ] SFB），25-37一个博尔顿猎人型试剂与生物分子的主要氨基酸残基的反应，是一个非常通用的的修复组为18架F -标签广泛的生物实体，其在体内的稳定性和高放射性标记的产量明显。经过与标签[18 F] SFB的，由此产生的[18 F] fluorobenzoylated生物分子在活体成像研究可以探讨潜在的PET示踪剂。1 [18 F] SFB在目前的文献描述radiosyntheses需要两个甚至三个反应堆和使用固相萃取（SPE）或高效液相色谱（HPLC）的多个纯化。这种漫长的过程，妨碍其日常生产和放射性标记的生物分子中的广泛应用。虽然几个模块辅助[18 F] SFB的合成已有报道，29-32，41-42，他们主要是基于复杂和漫长的程序，使用昂贵的市售放射化学盒（ 见表 1 ）。因此，进一步简化的放射合成[18 F] SFB使用一个低成本的设置将其适应一个自动化的过程十分有利。 在这里，我们报告一个简洁的准备[18 F] SFB的，基于一种简化的微波辅助合成一锅煮（ 图1）。我们的方法，不需要净化步骤或任何水试剂之间。此外，微波辐射，已在几个PET示踪剂的合成，38-41可以提供更高RCYs和更好的选择性比相应的热反应或他们提供类似的收益率在更短的反应时间 。38最重要的是，当标签的生物分子，节省的时间可以改行随后bioconjugation或PET显像步骤28,43新颖的改进[18 F] SFB的合成是两个方面：（1）无水脱保护战略的需要之间没有中间（S）的净化每个步骤和（2）微波辅助放转换实现快速，可靠的生产[18楼 ] SFB。

Watch this Scientific Journal Video about Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB at JoVE.com

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

一个轻便，一锅煮合成N -琥珀酰亚胺- 4 - [<sup&gt; 18</sup&gt; F] fluorobenzoate（[<sup&gt; 18</sup&gt; F] SFB）是基于非水，三步走放过程。利用微波加热，就可以完成整个过程在不到30分钟，或60分钟，进一步纯化制备高效液相色谱法。衰减校正（RCYs）放的产量分别为35-5％（N&gt; 30）。

微波辅助合成一锅 N -琥珀酰亚胺- 4 - [ 18 F] fluorobenzoate（[ 18 F] SFB）

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

microwave-assisted-one-pot-synthesis-n-succinimidyl-4

Research

JoVE Journal

Biology

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

12.9K 次观看.  University of California at Los Angeles. 一个轻便，一锅煮合成N -琥珀酰亚胺- 4 - [ 18 F] fluorobenzoate（[ 18 F] SFB）是基于非水，三步走放过程。利用微波加热，就可以完成整个过程在不到30分钟，或60分钟，进一步纯化制备高效液相色谱法。衰减校正（RCYs）放的产量分别为35-5％（N> 30）。

视频: Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

卯木反应的优化使用平行合成和自动化液体处理

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

科研

生物学

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

氧成像在生物系统中的磷光纳米探针的合成和校准

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

合成的<em&gt;在体内</em&gt; MRI检测细胞凋亡的探讨

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

Solid Phase Synthesis of a Functionalized Bis-Peptide Using &quot;Safety Catch&quot; Methodology

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

一个官能二肽的固相合成使用“安全锁扣”方法论

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

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

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

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

全球合成的RNA在单细胞水平以下的低氧分析

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

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

通过使用光稳定菁染料波长移的DNA杂交探针的合成

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

卡诺哈布迪蒂斯 伊利根斯新蛋白合成率评估

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...