这项研究由国家老化（NFN S93）由奥地利科学基金会（FWF），欧盟委员会综合项目MiMAGE和PROTEOMAGE，荷兰基因组计划研究网络/荷兰科学研究组织（NGI / NWO; 05040202，050 - 060-810恩查），欧盟资助网络的卓越寿命（FP6 036894），和基因组学创新研究计划（SenterNovem IGE01014和IGE5007）。

1。质粒的构建<ol><li>订购定制寡核苷酸编码为DGN（ACKNWFSSLSHFVIHL 11）和为dgnFS（HARTGSLACPTSSSICE）和结扎到质粒pEGFP-C1载体，以获得与DGN / dgnFS的（ 图1）的GFP的融合。 </li><li>通过PCR扩增的GFP-dgn和GFP-dgnFS的的编码序列，。根据协议pENTR定向TOPO克隆试剂盒，并继续与pLenti6/V5定向TOPO克隆试剂盒（ 图6）。 </li></ol> 2。病毒生产<ol><li>转染前一天（第1天）的种子，满分HEK 293FT细胞在T75烧瓶中，以便它们将是90-95％汇合的转染当天。 </li><li>在转染当天，在1.5毫升的DMEM稀释36μl的脂质体Lipofectamine 2000试剂，无血清和抗生素在一个15毫升鹘。另外15毫升猎鹰和稀释3微克pLenti6/V5携带互补的DNA构造或者GFP<html> DGN或GFP-dgnFS的的，2.5微克信封编码质粒pMD2.G和7.5微克载体骨干psPAX2的在1.5毫升无血清和抗生素的DMEM中。 5分钟后稀释的DNA与稀释的脂质体Lipofectamine 2000试剂相结合。 </li><li>孵育混合物20分钟，在室温下，以允许DNA-脂质体转染法络合物形式。 </li><li>的HEK 293FT细胞中取出介质，取代它仔细7毫升培养基（不含抗生素）和DNA-脂质体混合物的烧瓶和岩石，它来回混合（2天）。在湿润的5％CO 2培养箱中，将烧瓶在37℃下过夜孵育。 </li><li>改变介质的第二天（不含抗生素的10毫升培养基，第3天）。 </li><li> 48小时后（第5天）收获上清液，在300×g离心5分钟，在室温下离心，并通过0.45μm的PVDF过滤器过滤上清液。 </li></ol> 培养基- ​​DMEM（HEK 293FT） 帐篷“&gt; DMEM 10％FBS的 0.1毫米MEM NEAA 6 mM的L-谷氨酰胺 1毫米MEM丙酮酸钠 1％PEN链球菌（可选） 500微克/毫升遗​​传霉素（可选） 3。病毒浓度的聚乙二醇（PEG）沉淀<ol><li>加入一体积的聚乙二醇溶液（50mM的聚乙二醇，41 mM氯化钠，高压釜中，pH值= 7.2，PEG）4体积的上清液和在4℃下孵育2小时小心地混合每20-30分钟反相。 </li><li>自旋向下在1500 xg离心30分钟，在4°C应该可以看到一个白色的小球。 </li><li>吸取上清，在1500×g离心5分钟，并再次离心在4℃下吸出剩余的PEG解决方案。 </li><li>将沉淀重悬于培养基或磷酸盐缓冲盐水（PBS），通过移液向上和向下大力为20至30秒的涡流。作为一个指导原则使用T75烧瓶和分装到100μL500μL。病毒存放于-80℃。 </li></ol><ol><li>满分5×10 4 U2-OS细胞转染前一天的6孔板的各孔中的种子。 </li><li>在转染当天，除去培养基，并替换它由1毫升含有8微克/毫升聚凝胺（溴化己二甲铵溴化物）的DMEM中。浓缩病毒上清1:10和1:100稀释，并加入1μl一个好。对于更高的病毒浓度，用1微升，加入5μl和10μl浓缩病毒上清的剩余井。一个阱被留下作为阳性对照。 </li><li>翌日2ml的DMEM替换培养基。 </li><li>开始第二天的选择。应用10μg/ ml的杀稻瘟菌素各孔上。每隔一天更换抗生素的培养基。 </li><li>约6-7天后开始选择未转染的细胞都死了。对于染色洗细胞3次，用PBS和覆盖细胞，用结晶紫（10毫克/升结晶紫在20％乙醇），并在室温下孵育5-10分钟。吸液结晶紫（可以重复使用几次）和两次DDH 2 O冲洗井和干板。 </li><li>计数菌落并乘以1000和其对应的稀释（ 图7）。此过程可以计算转染单元（TU）的病毒/毫升。 </li></ol> 培养基- ​​DMEM（HFF-2/U2-OS） DMEM 10％FBS的 6mm的L-谷氨酰胺 笔链球菌1％ 5。人二倍体成纤维细胞转导（此步骤可用于任何类型的细胞。） <ol><li>种子从5×10 4人二倍体成纤维细胞（HDF）中的6孔板中的转导的前一天。 </li><li>使用多重感染两个一起聚凝胺8微克/毫升一毫升，共转导增强。 </li><li>改变介质的第二天。 </li><li>何时的细胞会合开始用10μg/ ml的杀稻瘟素的选择是在70-80％的。选择完成后（没有更多未转染的细胞死亡）的细胞就可以开始扩大，使细胞为实验做好准备。杀稻瘟素的慢性选择与允许生成稳定的细胞系过度GFP-DGN或GFP-dgnFS的融合蛋白，准备测量蛋白酶体活性。此过程保证独立的细胞类型的一个非常高的转染效率。 </li></ol> 6。通过流式细胞仪的测量<ol><li>种子从1×10 5个细胞（携带GFP-DGN或GFP-dgnFS的的）实验的前一天。 </li><li>处理对照细胞前3小时，与蛋白酶体抑制剂的测量， 例如 ，100微克/毫升LLNL。 </li><li>用PBS洗两次，用0.5毫升的胰蛋白酶收集细胞。要停止胰蛋白酶消化使用4.5毫升培养液中的细胞转移到15毫升猎鹰的。 </li><li>自旋吨细胞在300×g离心5分钟，在室温下。 </li><li>吸取培养基，重新悬浮在PBS中的细胞，并再次降速在​​300×g离心5分钟，在37℃下</li><li>吸取PBS，重新悬浮于400μlFACS缓冲液中（10毫摩尔的Hepes，140 mM氯化钠，2.5mM的CaCl 2的 ，pH值= 7.4），和细胞转移到FACS管。 </li><li>保持冰和测量样品的细胞通过流式细胞仪检测。细胞不应该长于30分钟被存储在4℃下</li></ol> 7。代表性的成果在GFP-DGN融合蛋白进行的序列，这是针对蛋白酶体，并因此，该蛋白质是立即退化;它对应于GFP荧光信号的减少。移码突变（GFP-dgnFS）进行该序列的突变版本的，是不被蛋白酶体降解，它会导致更高的绿色荧光。出于这些原因，年轻的与预期的高蛋白酶体活性的HDFS和转用GFP-分克n显示低（6％的阳性细胞）的荧光信号在两个流式细胞仪测量和在落射荧光（ 图2A和B，图3）。同一个HDFS转GFP-dgnFS的显示39.7％的阳性细胞。细胞的治疗与蛋白酶体抑制剂LLNL（N-乙酰基-L-亮氨酰-L-亮氨酰-L-norleucinal）提出的信号两者，GFP-dgn和的GFP-dgnFS细胞的（ 图2A和B中的62.9％的最大）。 以确定可能的老年人类皮肤样本的蛋白酶体活性的下降，从青年，中年和老年人的捐助者分离的皮肤成纤维细胞与GFP dgn和GFP-dgnFS的的感染，如上所述，培养前相同的通道数流量分析流式细胞仪。在这些实验中，一个鲜明的年轻个体之间的GFP信号增加（11.2±0.88％GFP阳性细胞）和中年捐助者（20.4±2.27％GFP阳性细胞，P = 0.003），说明已被观察到减少龙眼青梅中年捐助者7（ 图4）从得到的样品中的活性。在蛋白酶体活性的最古老的个体（ 图4）从分离的成纤维细胞中观察到没有进一步的减少。为GFP-dgnFS的是在所有情况下，荧光强度〜90％（数据未示出），这表明了高转染效率为三个不同的年龄组。 <img src="/files/ftp_upload/3327/3327fig1.jpg" alt="图1" /> 图1。在GFP-dgn和的GFP-dgnFS序列的显示是，GFP（绿色）的多个克隆位点的pEGFP-CL1载体（灰色）和的DGN / dgnFS序列（红色）的3&#39;-末端。 <img src="/files/ftp_upload/3327/3327fig2.jpg" alt="图2" /> 图2。分析GFP-DGN和GFP-dgnFS的的人二倍体成纤维细胞的流式细胞仪。 A.年轻的人包皮成纤维细胞（HFF-2）感染了慢病毒载体的绿色荧光蛋白（GFP）DGN基因或GFP-dgnFS（移码）构造，使用流式细胞仪（FACS粤语II，Becton Dickinson公司）的指示，分析GFP荧光。另有说明外，细胞也处理3h处理与蛋白酶体抑制剂N-乙酰基-L-亮氨酰-L-亮氨酰-L-norleucinal（LLNL）。未感染的细胞用作对照。实验一式三份进行。B.数据是三个独立实验的代表。这些数字反映的GFP阳性细胞的数量。 <a href="http://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/3327/3327fig3.jpg" alt="图3" /> 图3。被视为图2中的GFP-dgn和GFP-dgnFS的的HFF-2细胞的荧光显微镜分析。杨HFF-2。在感染后第9天，细胞呈V荧光相差显微镜isualized通过。 <img src="/files/ftp_upload/3327/3327fig4.jpg" alt="图4" /> 图4。在人体皮肤老化的蛋白酶体活性的变化。从9个不同的捐赠者在指定的年龄组的人包皮成纤维细胞的慢病毒结构编码GFP-DGN最低限度的扩大和感染。在感染后第9天，细胞，用流式细胞仪分析。数据上得到了每个年龄组的三个样本一式两份（±SE）。 <img src="/files/ftp_upload/3327/3327fig5.jpg" alt="图5" /> 图5。实验方法。自定义寡核苷酸DGN和dgnFS的克隆到pEGFP-C1的载体和病毒为每个使用HEK 293FT细胞的结构。确定病毒的滴度。将细胞与病毒转导和扩大。后优惠待遇细胞分析通过流式细胞仪检测荧光信号。 <img src="/files/ftp_upload/3327/3327fig6.jpg" alt="图6" /> 图6。 GFP-DGN序列的pLenti6/V5-DEST矢量地图的：GFP-DGN pLenti。的地图显示。缩写：PCMV（CMV启动子），GFP-DGN（序列的GFP-DGN），P SV40（SV40早期启动子），EM7（EM7子），稻瘟菌素（杀稻瘟抗性基因），ΔU3/ 3&#39;LTR（3&#39;LTR与删除U3区域），SV40，SV40多聚腺苷酸化信号（pA的），氨苄西林（氨苄青霉素抗性基因），pUC系毛利（pUC的原点），PRSV / 5&#39;LTR（RSV / 5&#39;LTR杂合启动子），Ψ（HIV-1Ψ包装信号），HIV-1 REV应答元件（RRE）。 <img src="/files/ftp_upload/3327/3327fig7.jpg" alt="图7" /> 图7。 U2-OS滴定板。U2-OS接种6孔板上，并与不同的病毒浓度（稀释1/100和1/10，1，5和10μl的共同转染的ncentrated病毒上清）。作为一个未转染对照（NT）。经过6-7天，将细胞染色，用结晶紫和在空气中干燥。

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没有利益冲突的声明。

用绿色荧光蛋白（GFP）作为底物泛素-蛋白酶体活性记者，发表于2000年12首次出版。从那时起，GFP已成为一种普遍的工具，可视化细胞的活动，特别是泛素 - 蛋白酶体的过程。若要监视在体内的泛素-蛋白酶体活性的转基因小鼠模型与基于GFP的记者已被引入13。额外另一种转基因小鼠模型的体内研究建立一个类似的不稳定degron的GFP记者作为本出版物中的14。 <p...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> PEGFP-C1载体</td><td> BD生物科学Clontech公司</td><td> 6084-1 </td></tr><tr><td> pENTR定向TOPO克隆试剂盒</td><td> Invitrogen公司</td><td> K2400-20 </td></tr><tr><td> pLenti6/V5定向TOPO克隆试剂盒</td><td> Invitrogen公司</td><td> V496-10 </td></tr><tr><td>脂质体转染试剂</td><td> Invitrogen公司</td><td> 11668019 </td></tr><tr><td> DMEM </td><td>西格玛</td><td> D5546 </td></tr><tr><td> PVDF过滤器（Rotilabo-Spritzenfilter） </td><td>罗斯</td><td> P667.1 </td></tr><tr><td>聚乙二醇</td><td>西格玛</td><td> P2139 </td></tr><tr><td>氯化钠</td><td>默克</td><td> 1.06404.1000 </td></tr><tr><td> Dulbecco磷酸盐缓冲贝东北1X（PBS） </td><td> Invitrogen公司</td><td> 14190 </td></tr><tr><td>溴化己二甲铵溴化</td><td>西格玛</td><td> 10,768-9 </td></tr><tr><td>杀稻瘟</td><td> Invitrogen公司</td><td> R21001 </td></tr><tr><td>结晶紫</td><td>西格玛</td><td> C3886 </td></tr><tr><td> FACS管</td><td> BD Biosciences公司</td><td></td></tr><tr><td>青霉素链霉素（笔链球菌） </td><td> Invitrogen公司</td><td> 15140130 </td></tr><tr><td> L-谷氨酰胺200mM的</td><td> Invitrogen公司</td><td> 25030024 </td></tr><tr><td>胎牛血清（FBS） </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM非必需氨基酸（NEAA）100X </td><td> Invitrogen公司</td><td> 11140035 </td></tr><tr><td> MEM丙酮酸钠100毫米</td><td> Invitrogen公司</td><td> 11360039 </td></tr><tr><td> D-（+）葡萄糖（45％） </td><td>西格玛</td><td> G8769 </td></tr><tr><td>标记基因</td><td> Invitrogen公司</td><td> 11811023 </td></tr><tr><td>氯化钙</td><td>默克</td><td> C5080 </td></tr><tr><td> HEPES </td><td>西格玛</td><td> H3375 </td></tr><tr><td>胰蛋白酶-EDTA（0.05％） </td><td> Invitrogen公司</td><td> 25300054 </td></tr></tbody></table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

蛋白酶是异常的，错误折叠的，损坏或氧化蛋白1，2中的蛋白水解降解的主要涉及的胞内细胞器。蛋白酶体活性的维护被牵连了许多关键的细胞过程，如细 ​​胞的应激反应，细胞周期调控和细胞分化或免疫系统的反应5。已与泛素-蛋白酶体系统的功能障碍，肿瘤，神经退行性疾病4，6的发展。此外，在蛋白酶体活性的减少被发现作为一个功能的细胞衰老和有机体的老化7，8，9，10。在这里，我们提出了一种方法来衡量使用GFP-的DGN融合蛋白在活细胞的泛素 - 蛋白酶体的活性。至能够监视泛素 - 蛋白酶体活性的活的原代细胞，互补的DNA构建体编码的绿色荧光蛋白（GFP）-DGN融合蛋白（GFP-DGN，不稳定）和携带的移码突变的变体（GFP-dgnFS的，稳定11）被插入在慢病毒表达载体。我们更喜欢这种技术比传统的转染技术，因为它保证了一个非常高的转染效率的细胞类型或捐赠者的年龄无关。可以使用显示在GFP-dgnFS（稳定）蛋白和蛋白酶体抑制剂的存在或不存在中的不稳定的蛋白（GFP-DGN）的荧光之间的差异，来估计在每个特定的细胞株的泛素 - 蛋白酶体活性。这些差异可以通过荧光显微镜监测，也可以通过流式细胞仪检测。

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

监控活细胞的泛素 - 蛋白酶体活性的方法。一个degron不稳定的绿色荧光蛋白（GFP-DGN）和一个稳定的的GFP-dgnFS融合蛋白的生成和使用慢病毒表达载体导入细胞。这种技术允许产生稳定的GFP-dgn/GFP-dgnFS表达细胞株中，泛素 - 蛋白酶体活性可以很容易地使用的反射荧光或流式细胞术评估。

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

monitoring-ubiquitin-proteasome-activity-living-cells-using-degron

Research

JoVE Journal

Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

16.6K 次观看.  Institute for Biomedical Aging Research. 监控活细胞的泛素 - 蛋白酶体活性的方法。一个degron不稳定的绿色荧光蛋白（GFP-DGN）和一个稳定的的GFP-dgnFS融合蛋白的生成和使用慢病毒表达载体导入细胞。这种技术允许产生稳定的GFP-dgn/GFP-dgnFS表达细胞株中，泛素 - 蛋白酶体活性可以很容易地使用的反射荧光或流式细胞术评估。

视频: Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

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

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

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

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...