We thank the Australian Research Council (ARC) for ARC Future Fellowship (FT120100101) and ARC Centre of Excellence CE140100036) grants to P.T. and the Mark Wainwright Analytical Centre at UNSW for access to mass spectrometry and NMR facilities.

注意: 如图1下面协议设计用于蛋白质-染料生物共轭的合成，可用于与含有游离表面半胱氨酸蛋白马来酰亚胺的反应的一般协议，与笔记插入在适用与膜蛋白来协助。生物共轭物，蛋白质类聚合物生物缀合物，以及合成的蛋白二聚体（蛋白质 - 蛋白质）生物缀合物。在这种特殊情况下，所述蛋白质异1色素c具有可用下反应一个表面半胱氨酸残基，它允许发生高度特异性的标记。如果感兴趣的蛋白具有多个半胱氨酸残基，相同的协议适用于，尽管有特异性和产品均匀性的损失。化学定位表面赖氨酸残基，用N- -hydroxysuccinimidyl酯或异硫氰酸酯，可以是如果不需要的特异性的简单的方法。 <img alt="图1" src="/files/ftp_upload/54157/54157fig1.jpg" /> <strong&gt;图1. 生物缀反应流程。作为一个例子的情况下，光收集，基于钌天线分子将通过迈克尔加成在基于钌天线分子的侧马来酰亚胺和暴露的半胱氨酸残基附着到细胞色素C（CYS102）上的蛋白。在细胞色素C面的红色区域表示血红素组。 <a href="https://www.jove.com/files/ftp_upload/54157/54157fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1，细胞色素C的分离纯化注意:此步骤是并不适用于所有的蛋白质。但是，重要的是要知道，从商业供应商获得的蛋白质可含有可能需要通过进一步纯化13去除其他不希望的蛋白同种型。 <ol><li>在1升的超纯水以溶解2.4克磷酸二氢钠（男女 = 120克摩尔-1）公关epare含有20mM的NaH 2 PO 4缓冲溶液。调节pH用1M NaOH至pH 7。 注意:在这个协议中使用的磷酸盐缓冲剂应新鲜每天制备，并通过在使用前用0.2μm的纤维素膜过滤器过滤。 </li><li>溶解在500毫升20毫摩尔的NaH 2 PO 4缓冲29.22克氯化钠（M W =58.44克摩尔-1）做出的20mM的NaH 2 PO 4和1M NaCl的缓冲液中。这是为在步骤1.6）中纯化的洗脱缓冲液。 </li><li>在6ml pH为7的20mM磷酸盐缓冲液中溶解12.0毫克冻干的细胞色素C（色素C）的。 </li><li>分别在95.3微升超纯水溶解14.7毫克二硫苏糖醇（DTT中，M W = 154.25克/摩尔）以制备1M的原液。 注意:DTT原液应新鲜制备，如该试剂易受在水溶液中的氧化失活。 </li><li>果仁ETTE 60微升的1M DTT溶液到该蛋白质溶液中，以减少色素C。溶液的颜色从暗红混合时变为淡红色。 </li><li>注入到任何色谱介质之前过滤通过0.22μm低蛋白结合的PVDF针筒过滤器还原蛋白溶液。 </li><li>附加注射环和一个快速蛋白质液相色谱（FPLC）仪器的UV-Vis检测之间的3.3毫升强阳离子交换柱。 </li><li>用超纯水的3倍柱体积，随后的20mM pH7的磷酸盐缓冲液3倍柱体积平衡色谱柱，使用1毫升/分钟的流速。 </li><li>负载1毫升降低原油色素C的进样环，并开始从328梯度方法- 450毫米氯化钠，超过5柱体积。监视UV-Vis检测的280纳米和410纳米的通道，并收集最大峰。 </li><li>增加盐浓度至1M为2倍柱体积洗脱异2细胞色素权证 ，并且列被刷新后，用20mM磷酸缓冲液2倍柱体积注入下一个粗等分试样之前重新平衡色谱柱。 </li><li>直到所有的粗蛋白已经被净化重复步骤1.9-1.10。 </li><li>池的iso-1含有级分，并用3.5 kDa的分子量截留（MWCO）旋转过滤器，并在3000×g下离心集中起来。 </li><li>加载的浓缩蛋白质进入3.5 kDa的截留分子量透析磁带和透析对超纯水过夜，水2的变化。 </li><li>通过采取10微升，其用超纯水稀释至100μl，并采取的吸收光谱确定的纯度，浓缩蛋白溶液的浓度。使用低容量，100微升石英比色皿，以获得蛋白质的吸收光谱。通常情况下，未稀释的蛋白质浓度是50量级 - 100微米。 <ol><li>使用细胞色素C的特征为410nm的峰值量化concentratioN使用比尔-朗伯定律97.6 -1毫米-1的摩尔吸光值: A =ε×C×ι 其中A是吸光度，ε是摩尔吸光系数，c为以mM的浓度，ι是试管的在厘米13的路径长度。 </li></ol></li><li>此时，划分蛋白质在1ml等份并于-20℃保持冷冻直到需要它们。色素C可以冷冻和没有结构或功能丧失解冻，但重复周期将开始使蛋白质变性。 注:蛋白质忍受冰冻程度不同。例如，绿色荧光蛋白5,9-不应被冻结，而不是保存在冰箱中。 </li></ol> 2，细胞色素C生物共轭物的合成<ol><li>溶解0.9毫克钌（Ⅱ）bisterpyridine maleimIDE（钌（Ⅱ）（TPY）2 -马来酰亚胺，0.975微摩尔，6当量）在600微升的乙腈中。 注意:如果使用的马来​​酰亚胺可溶于水，制备在水代替乙腈原液。为马来酰亚胺可溶于反应缓冲液是重要的。二甲亚砜，N，N-二甲基甲酰胺和乙腈中所有常用的辅助剂，以帮助在低分子量12，常疏水马来酰亚胺反应物的溶解。 </li><li>制备含有100mM磷酸盐缓冲液和100mM乙二胺四乙酸（EDTA中，M W =292.24克/摩尔），其中，当稀释至20mM的pH为7.加入固体氢氧化钠直到EDTA溶解（几克典型地为缓冲溶液）并调节pH至7。 </li><li>溶解2.87毫克三（2-羧乙基）膦盐酸盐（TCEP 15中，M W = 286.65克/摩尔）在1ml超纯水中，以制备10mM储备溶液。这一步是进口蚂蚁，以确保蛋白质上半胱氨酸完全成马来酰亚胺偶联15之前降低。 注意:TCEP原液应新鲜每个反应来制备，因为这种试剂是容易在水溶液中的氧化失活。 </li><li>结合11.4毫升超纯水与50毫升的塑料管3毫升100mM磷酸/ EDTA储液。当与蛋白质和马来酰亚胺储备溶液进一步稀释缓冲液浓度为20毫摩尔。 注意:如果被标记的蛋白是跨膜蛋白，确保一个合适的洗涤剂加入到缓冲以溶解蛋白质。如果不使用洗涤剂的膜蛋白可以慢慢沉淀，这将不利地影响反应产率。使用该洗涤剂中的所有随后的纯化步骤。 </li><li>添加0.15微摩尔（3毫升50微米的溶液，1当量）纯化色素C的对磷酸EDTA缓冲液。 </li><li>加入7.5微升的TCEP股票SOLUT的离子（0.075微摩尔​​，0.5当量）的蛋白质溶液和休假搅拌5分钟，以减少可能由于半胱氨酸氧化二聚化的任何蛋白质。 注意:不要添加TCEP如果感兴趣的蛋白质不容易二聚化。通过非还原条件​​下运行的蛋白质凝胶检查。 </li><li>添加600μl的钌（II）（TPY）2 -马来酰亚胺到细胞色素C的减少，缓冲溶液的乙腈溶液，并留下该反应混合物搅拌，在黑暗中，在室温，24小时。 </li><li>集中使用3.5 kDa的MWCO自旋过滤器将反应混合物，离心以3,000 xg离心，用新鲜的20 mM磷酸盐缓冲液重复2-3次，直到滤液变清。纯化的下一阶段之前，除去从反应混合物中尽可能多的未反应的马来酰亚胺。 注意:在这一点上，将粗生物结合混合物可以储存在冰箱内，在黑暗中。它以消除CENTR未反应的马来酰亚胺染料是重要的ifuge储存前透析作为马来酰亚胺可以在蛋白质的表面上的赖氨酸残基慢慢和非特异性反应。 </li></ol> 3.细胞色素C生物共轭物的分离纯化<ol><li>溶解2.4克磷酸二氢钠和在1升超纯水29.22克氯化钠以制备含有20mM的NaH 2 PO 4和0.5M NaCl的缓冲液。这是用于固定化金属亲和层析（IMAC）纯化的运行缓冲液。 </li><li>溶解在500毫升20毫摩尔的NaH 2 PO 4和0.5M NaCl的缓冲液为17克咪唑（M W =68.077克摩尔-1）。这是对IMAC纯化的洗脱缓冲液。 </li><li>调整两个缓冲液的pH到使用1M的NaOH或HCl pH为7，并在FPLC使用前通过0.22μm的再生纤维素膜过滤。 </li><li>由于购买的IMAC柱出厂时没有加载到山坳任何金属离子UMN，通过用3ml的超纯水，将3ml 100mM的乙酸镍溶液，和6ml的超纯水洗涤柱制备了镍离子为基础的纯化塔。如果该列没有被立即使用冲洗通过3毫升20％乙醇和列存储在冰箱，以防止细菌的生长。 </li><li>附加的Ni 2+ -loaded1毫升IMAC柱的FPLC注入环和UV-Vis检测器之间。 </li><li>用20mM磷酸的3倍柱体积，用0.5毫升/分钟的流速0.5M NaCl的缓冲液平衡该柱。 </li><li>负载100微升对Ni 2+ IMAC柱粗反应混合物（通过0.22μm针筒过滤器过滤）的。用运行缓冲液以洗脱未反应的色素C的3倍柱体积，随后从0咪唑梯度的125mM以上3倍柱体积洗涤柱以洗脱钌bisterpyridine-色素c生物共轭（钌（Ⅱ）-cyt 三 ） 。 </li><li>洗净的Column用5柱体积的250 mM咪唑缓冲液，然后用20 mM磷酸，0.5M NaCl和重复步骤3.7，直到所有的粗生物共轭已被纯化重新平衡色谱柱。 </li><li>凝聚生物共轭级分，并用3.5 kDa的MWCO旋转过滤器，以3000×g的离心分离集中起来。 </li><li>装载集中到生物共轭物3.5 kDa的截留分子量透析磁带和透析对超纯水过夜，水2的变化。 </li><li>确定由UV-VIS纯，浓缩生物共轭溶液的浓度，使用相同的摩尔吸光值作为异1色素c（97.6毫-1厘米-1）在410纳米。通常情况下，生物共轭浓度为50量级 - 100微米。 </li><li>划分成生物共轭物25微升等分，并在-20°C保持冷冻直到需要。 </li></ol> 4.表征细胞色素C的生物共轭<ol><li> 阻止通过MALDI-TOF质谱生物共轭质量mination <ol><li>溶解10毫克的咖啡酸的在1毫升乙腈/水/三氟乙酸溶液（80:20:0.1，V / V / V）。 </li><li>稀释5μl的浓缩的蛋白质溶液与5微升的咖啡酸​​溶液。 </li><li>点0.5微升在MALDI靶盘咖啡酸溶液，并使溶液干燥。 </li><li>现货0.5微升这一咖啡酸点上方的采样/矩阵解决方案，允许当场晾干。发现在此之上，以"三明治"基质的层间的试样另一0.5微升样品基质的，并晾干。 </li><li>使用合适的仪器设置的蛋白质16取得以线性模式质谱。 </li></ol></li><li> 通过凝胶电泳生物缀合物的调查 <ol><li>制备10微升每个蛋白质样品以稀释蛋白质样品与预混合十二烷基硫酸锂（LDS，pH值8.4）缓冲液（4次）T为运行OA的每孔约20微克最终浓度。对于需要还原条件下水井，加入1微升500二硫苏糖醇（DTT）的还原剂（10X）的。 </li><li>热样品在70℃下10分钟。 </li><li>加50预混毫升的1M MES，1M Tris碱，2％SDS，20毫摩尔EDTA（pH值7.7）从商业来源至950毫升的超纯水的运行缓冲液混合物（20倍），以制备凝胶电泳缓冲液。 </li><li>从凝胶中去除塑料梳子，并放置在凝胶运行槽中的凝胶。使孔覆盖有缓冲填充凝胶电泳缓冲液的罐。 </li><li>装载样品仔细到预制12％双Tris，1毫米，使用长枪头10孔凝胶，以协助装载。加载预染10蛋白质分子量标记物（3-188 kDa的）到凝胶的一个中间孔以有助于分析。 </li><li>运行在200伏的恒定电压的凝胶35分钟。 </li><li>染色用2小时商业考马斯蓝溶液的凝胶，并用ultrap洗URE水中48小时。 </li></ol></li><li> 生物共轭纯度通过UV-Vis光谱测定 <ol><li>制备120微升钌（Ⅱ）（TPY）2 -马来酰亚胺，异1细胞色素c和 ​​钌（II）-cyt 的c为5μM的解决方案。 </li><li>测量仅含有超纯水的石英比色皿的基线频谱，从250纳米到650纳米。 </li><li>测量每个元件的光谱，确保了反应杯漂洗和每次测量之间干燥。 </li><li>绘制每个部件的吸光度作为波长的函数，并且与最终产品相比较的原料的线性总和，以确定是否一个1:钌（II）（TPY）2 -马来酰亚胺来色素C的1比已反应。 </li></ol></li></ol>

<ol>
	<li>Griffin, B. A., Adams, S. R., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+covalent+labeling+of+recombinant+protein+molecules+inside+live+cells.">Specific covalent labeling of recombinant protein molecules inside live cells.</a> Science. 281, 269-272 (1998).</li><li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+chemistry:+Fishing+for+selectivity+in+a+sea+of+functionality.">Bioorthogonal chemistry: Fishing for selectivity in a sea of functionality.</a> Angew. Chem. Int. Ed. 48, 6974-6998 (2009).</li><li>Lyon, R. P., Meyer, D. L., Setter, J. R., Senter, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+of+anticancer+drugs+through+endogenous+monoclonal+antibody+cysteine+residues.">Conjugation of anticancer drugs through endogenous monoclonal antibody cysteine residues.</a> Meth. Enzymol. 502, 123-138 (2012).</li><li>Natarajan, A., Xiong, C. Y., Albrecht, H., DeNardo, G. L., DeNardo, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+site-specific+ScFv+PEGylation+for+tumor-targeting+pharmaceuticals.">Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals.</a> Bioconjug. Chem. 16, 113-121 (2005).</li><li>Hvasanov, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-Pot+Synthesis+of+High+Molecular+Weight+Synthetic+Heteroprotein+Dimers+Driven+by+Charge+Complementarity+Electrostatic+Interactions.">One-Pot Synthesis of High Molecular Weight Synthetic Heteroprotein Dimers Driven by Charge Complementarity Electrostatic Interactions.</a> J. Org. Chem. 79, 9594-9602 (2014).</li><li>Thordarson, P., Le Droumaguet, B., Velonia, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-defined+protein-polymer+conjugates--synthesis+and+potential+applications.">Well-defined protein-polymer conjugates--synthesis and potential applications.</a> Appl. Microbiol. Biotechnol. 73, 243-254 (2006).</li><li>Lutz, J. F., B&#246;rner, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+trends+in+polymer+bioconjugates+design.">Modern trends in polymer bioconjugates design.</a> Prog. Polym. Sci. 33, 1-39 (2008).</li><li>Nicolas, J., Mura, S., Brambilla, D., Mackiewicz, N., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+functionalization+strategies+and+biomedical+applications+of+targeted+biodegradable/biocompatible+polymer-based+nanocarriers+for+drug+delivery.">Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery.</a> Chem. Soc. Rev. 42, 1147-1235 (2013).</li><li>Wong, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersomes+Prepared+from+Thermoresponsive+Fluorescent+Protein-Polymer+Bioconjugates:+Capture+of+and+Report+on+Drug+and+Protein+Payloads.">Polymersomes Prepared from Thermoresponsive Fluorescent Protein-Polymer Bioconjugates: Capture of and Report on Drug and Protein Payloads.</a> Angew. Chem. Int. Ed. , 5317-5322 (2015).</li><li>Hermanson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. , (2013).</li><li>Li, J., Xu, Q., Cortes, D. M., Perozo, E., Laskey, A., Karlin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactions+of+cysteines+substituted+in+the+amphipathic+N-terminal+tail+of+a+bacterial+potassium+channel+with+hydrophilic+and+hydrophobic+maleimides.">Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides.</a> Proc. Natl. Acad. Sci. U.S.A. 99 (18), 11605-11610 (2002).</li><li>Peterson, J. R., Smith, T. A., Thordarson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+room+temperature+photo-induced+electron+transfer+in+biologically+active+bis(terpyridine)ruthenium(II)-cytochrome+c+bioconjugates+and+the+effect+of+solvents+on+the+bioconjugation+of+cytochrome+c.">Synthesis and room temperature photo-induced electron transfer in biologically active bis(terpyridine)ruthenium(II)-cytochrome c bioconjugates and the effect of solvents on the bioconjugation of cytochrome c.</a> Org. Biomol. Chem. 8, 151-162 (2010).</li><li>Borges, C. R., Sherma, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+the+Analysis+of+Cysteine+Sulfhydryls+and+Oxidative+Protein+Folding.">Techniques for the Analysis of Cysteine Sulfhydryls and Oxidative Protein Folding.</a> Antioxid. Redox Signal. (3), 1-21 (2014).</li><li>Peterson, J. R., Thordarson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+the+purification+of+terpyridine-cytochrome+c+bioconjugates.">Optimising the purification of terpyridine-cytochrome c bioconjugates.</a> Chiang Mai J. Sci. 36 (2), 236-246 (2009).</li><li>Hvasanov, D., Mason, A. F., Goldstein, D. C., Bhadbhade, M., Thordarson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+the+synthesis,+polymer+membrane+encapsulation+and+photoreduction+performance+of+Ru(II)-+and+Ir(III)-bis(terpyridine)+cytochrome+c+bioconjugates.">Optimising the synthesis, polymer membrane encapsulation and photoreduction performance of Ru(II)- and Ir(III)-bis(terpyridine) cytochrome c bioconjugates.</a> Org. Biomol. Chem. 11 (28), 4602-4612 (2013).</li><li>Signor, L., Boeri Erba, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+Laser+Desorption/Ionization+Time+of+Flight+(MALDI-TOF)+Mass+Spectrometric+Analysis+of+Intact+Proteins+Larger+than+100+kDa.">Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa.</a> J. Vis. Exp. , e50635 (2013).</li><li>Foucher, M., Verdi&#232;re, J., Lederer, F., Slonimski, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+presence+of+a+non-trimethylated+iso-1+cytochrome+c+in+a+wild-type+strain+of+Saccharomyces+cerevisiae).">On the presence of a non-trimethylated iso-1 cytochrome c in a wild-type strain of Saccharomyces cerevisiae).</a> Eur. J. Biochem. 31, 139-143 (1972).</li><li>M&#252;ller, M., Azzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+beef+heart+cytochrome+oxidase+subunit+III+with+eosin-5-maleimide.">Selective labeling of beef heart cytochrome oxidase subunit III with eosin-5-maleimide.</a> FEBS Lett. 184 (1), 110-114 (1985).</li><li>Shen, B. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+site+modulates+the+in+vivo+stability+and+therapeutic+activity+of+antibody-drug+conjugates.">Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.</a> Nat. Biotechnol. 30 (2), 184-189 (2012).</li></ol>

The authors have nothing to disclose.

一个生物结合前的原料纯化是非常重要的。从商业的重组来源得到的蛋白质常含有目的蛋白质，其可具有不同的表面化学和反应的其它同种型。例如，在所描述的生物结合，市售细胞色素C包含两个异1和异2色素C 12,14,17的混合物。异2和异1形式的细胞色素c在很大程度上是同源的，与主要的区别是异1细胞色素C的C-末端附近的一个游离半胱氨酸残基的存在?...

生物缀涉及共价连接一种生物分子与另一个或同一个合成的分子，例如染料，药物或聚合物。蛋白质的生物耦合方法现在广泛应用于许多化学，生物和纳米技术研究小组使用，应用范围从荧光染料标记1,2，使蛋白质（抗体）-prodrugs 3（抗体偶联药物- ADC）的蛋白二聚体4,5的合成通过在纳米8和系统化学9中使用的自组装蛋白-聚合物杂种6,7。 用于生物结合化学的特异性，而并非总是关键的，是对大多数功能性蛋白质生物共轭物极为重要，以便不与目标蛋白的活性位点干扰。提供了理想生物缀反应需要满足若干标准，包括:感兴趣的蛋白质上ⅰ）靶向稀有或独特的位点，ⅱ）是有选择性的实现这一目标，ⅲ）非变性条件下进行，以避免蛋白质折叠和iv）是高产作为目标蛋白质通常只适用于子毫摩尔浓度。马来酰亚胺-半胱氨酸迈克尔加成接近满足所有这些标准，并具有该原因长权利生物共轭化学10的字段的特殊状态。这是因为，ⅰ）在其表面上仅含有一个半胱氨酸残基的许多蛋白质​​可以遗传工程那里，ⅱ）在正确的pH值的反应是朝着半胱氨酸高度选择性，ⅲ）它可顺利地进行在水性缓冲液和iv）它是非常快的与马来酰亚胺的二阶速率常数报告超过5000 M -1秒-1在某些情况下11含有半胱氨酸的蛋白质。提供感兴趣的蛋白质可以容忍有机的一个小的（≈5-10％）量的共溶剂12，几乎任何马来酰亚胺官能化的染料，婆lymer，表面或另一种蛋白可以链接到蛋白质。此外，马来酰亚胺是对蛋白质比碘乙酰胺，这是更容易在升高的​​pH值的其他亲核体进行反应的半胱氨酸更具体;比这需要在酸性pH值被保持，以防止二硫键交换13基于二硫键缀合更稳定。 此处我们报告马来酰亚胺官能化的分子的缀合的通用协议包含使用钌（Ⅱ）基的发色团和氧化还原蛋白细胞色素C作为例子之间的反应的单个半胱氨酸残基的蛋白质。这个协议同样适用于含有可接近表面的半胱氨酸残基和相应的马来酰亚胺官能化的靶大多数其他蛋白，无论是另一种蛋白，荧光染料，发色团或合成聚合物。

<table border="0" cellpadding="0" cellspacing="0" width="913">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium dihydrogen phosphate</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">71496</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium hydroxide</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">71691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium chloride</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">73575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">cytochrome c, from saccaromyces cerevisiae</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">C2436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">dithiothreitol</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">TSKgel SP-5PW</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">Tosoh SP-5PW, 07161</td>
			<td>3.3 mL strong cation exchange column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15&#160;</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;">UFC900308</td>
			<td>3.5 kDa spin filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide-A-Lyzer mini dialysis units</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">66333</td>
			<td>3.5 kDa dialysis cassetes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ru(II) bisterpyridine maleimide</td>
			<td style="width:196px;">Lab made</td>
			<td style="width:151px;"></td>
			<td>see ref (14)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">acetonitrile</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">A3396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">ethylenediaminetetraacetic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">03609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tris(2-carboxyethyl)phosphine hydrochloride&#160;</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">93284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">imidazole</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">56749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">nickel acetate</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">244066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">AcroSep IMAC Hypercell column</td>
			<td style="width:196px;">Pall</td>
			<td style="width:151px;">via VWR: 569-1008</td>
			<td>1 mL IMAC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.2 micron cellulose membrane filter</td>
			<td style="width:196px;">Whatman</td>
			<td style="width:151px;">Z697958</td>
			<td>47 mm filter for buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.2 micron PVDF membrane filter</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;">SLGV013SL</td>
			<td>syringe filters for proteins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">hydrochloric acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">84426</td>
			<td>extremely corrosive! Use caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">caffeic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">60018</td>
			<td>MALDI matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">trifluoroacetic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">91707</td>
			<td>extremely corrosive! Use caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">LC6060</td>
			<td>Coomassie blue solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE Novex 12% Bis-Tris Gel</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0342BOX</td>
			<td>precast protein gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">SeeBlue Plus2 Pre-stained Protein Standard</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">LC5925</td>
			<td>premade protein ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE LDS Sample Buffer (4X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0008</td>
			<td>premade gel sample buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE Sample Reducing Agent (10X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0004</td>
			<td>premade gel reducing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE MES SDS Running Buffer (20X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0002</td>
			<td>premade gel running buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Voyager DE STR MALDI reflectron TOF MS</td>
			<td style="width:196px;">Applied Biosystems</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Acta FPLC</td>
			<td style="width:196px;">GE</td>
			<td style="width:151px;"></td>
			<td>Fast Protein Liquid Chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Cary 50 Bio Spectrophotometer</td>
			<td style="width:196px;">Varian-Agilent</td>
			<td style="width:151px;"></td>
			<td>UV-Vis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Milli-Q ultrapure water dispenser</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;"></td>
			<td>ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Low volume UV-Vis Cuvette</td>
			<td style="width:196px;">Hellma</td>
			<td style="width:151px;">105-201-15-40</td>
			<td>100 microliter cuvette</td>
		</tr>
	</tbody>
</table>

synthesis of protein bioconjugates via cysteine-maleimide chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

生物缀合物的合成通过三种主要方法证实:飞行质谱（MALDI-TOF MS），聚丙烯酰胺凝胶电泳的基质辅助激光解吸电离时间，和紫外可见（UV-VIS）光谱， 如图2所示，图3和4。对应于所附小分子的质量的质量增加，并且缺乏未反应蛋白质的演示钌（Ⅱ）的成功的共价键（万吨）2 -马来酰亚胺到细胞色素c和 ​​生物共轭的随后?...

Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

该协议细节对于含有蛋白质马来酰亚胺，包括试剂纯化，反应条件，生物共轭纯化和生物共轭表征半胱氨酸的生物结合所需的重要步骤。

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

synthesis-of-protein-bioconjugates-via-cysteine-maleimide-chemistry

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Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Chemical Applications of Statistical Analyses

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38.2K 次观看.  The University of New South Wales. 该协议细节对于含有蛋白质马来酰亚胺，包括试剂纯化，反应条件，生物共轭纯化和生物共轭表征半胱氨酸的生物结合所需的重要步骤。 

视频: Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides (CHIPS)

The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order to streamline synthesis by saving steps, time and material compared to conventional methods that require the introduction and removal of activating or directing groups. Therefore, the functionalization of C-H bonds is also attractive for green chemistry. Under oxidative conditions, two C-H bonds or one C-H and one heteroatom-H bond can be transformed to C-C and C-heteroatom bonds, respectively. Often these oxidative coupling reactions require synthetic oxidants, expensive catalysts or high temperatures. Here, we describe a two-step procedure to functionalize indole derivatives, more specifically tetrahydrocarbazoles, by C-H amination using only elemental oxygen as oxidant. The reaction uses the principle of C-H functionalization via Intermediate PeroxideS (CHIPS). In the first step, a hydroperoxide is generated oxidatively using visible light, a photosensitizer and elemental oxygen. In the second step, the N-nucleophile, an aniline, is introduced by Br&#248;nsted-acid catalyzed activation of the hydroperoxide leaving group. The products of the first and second step often precipitate and can be conveniently filtered off. The synthesis of a biologically active compound is shown.

Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides CHIPS

A two-step procedure for the synthesis of pharmaceutically active indole-derivatives by C-H functionalization with anilines is described, using photo- and Br&#248;nsted acid catalysis.

通过中级过氧化物的抗病毒四氢咔唑衍生物的合成光化学和酸催化CH官能（筹）

The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order ...

科研

化学

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

功能化的合成与表征金属 - 有机骨架

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthesis and Reaction Chemistry of Nanosize Monosodium Titanate

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the material with Au(III) ions. The synthesis method was derived from a sol-gel process used to produce micron-sized monosodium titanate (MST), with several key modifications, including altering reagent concentrations, omitting a particle seed step, and introducing a non-ionic surfactant to facilitate control of particle formation and growth. The resultant nMST material exhibits spherical-shaped particle morphology with a monodisperse distribution of particle diameters in the range from 100 to 150&#160;nm. The nMST material was found to have a Brunauer-Emmett-Teller (BET) surface area of 285 m2g-1, which is more than an order of magnitude higher than the micron-sized MST. The isoelectric point of the nMST measured 3.34 pH units, which is a pH unit lower than that measured for the micron-size MST. The nMST material was found to serve as an effective ion exchanger under weakly acidic conditions for the preparation of an Au(III)-exchange nanotitanate. In addition, the formation of the corresponding peroxotitanate was demonstrated by reaction of the nMST with hydrogen peroxide.

The surfactant mediated sol-gel synthesis of nanosized monosodium titanate is described, along with preparation of the corresponding peroxide modified material. An ion-exchange reaction with Au(III) is also presented.

纳米尺寸钛酸二氢钠的合成和化学反应

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the ...

The Synthesis of [Sn10(Si(SiMe3)3)4]2- Using a Metastable Sn(I) Halide Solution Synthesized via a Co-condensation Technique

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a sterically demanding ligand, has increased in recent years. The metastable Sn(I) halide is synthesized at&#160;&#34;outer space conditions&#34; via the preparative co-condensation technique. Thereby, the subhalide is synthesized in an oven at high temperatures, around 1,300 &#176;C, and at reduced pressure by the reaction of elemental tin with hydrogen halide gas (e.g., HCl). The subhalide (e.g., SnCl) is trapped within a matrix of an inert solvent, like toluene at -196 &#176;C. Heating the solid matrix to -78 &#176;C gives a metastable solution of the subhalide. The metastable subhalide solution is highly reactive but can be stored at -78 &#176;C for several weeks. On heating the solution to room temperature, a disproportionation reaction occurs, leading to elemental tin and the corresponding dihalide. By applying bulky ligands like Si(SiMe3)3, the intermediate metalloid cluster compounds can be trapped before complete disproportionation to elemental tin. Hence, the reaction of a metastable Sn(I)Cl solution with Li-Si(SiMe3)3 gives [Sn10(Si(SiMe3)3)4]2- 1 as black crystals in high yield. 1 is formed via a complex reaction sequence including salt metathesis, disproportionation, and degradation of larger clusters. Further, 1 can be analyzed by various methods like NMR or single crystal X-ray structure analysis.

The Synthesis of [Sn10SiSiMe334]2- Using a Metastable SnI Halide Solution Synthesized via a Co-condensation Technique

The disproportionation reaction of a metastable Sn(I) chloride solution, obtained via the preparative co-condensation technique, is used for the synthesis of a metalloid tin cluster compound.

的合成[锡<sub&gt; 10</sub&gt;（SI（SIME<sub&gt; 3</sub&gt;）<sub&gt; 3</sub&gt;）<sub&gt; 4</sub&gt;]<sup&gt; 2</sup<sup&gt; -</sup&gt;使用亚稳锡（I）卤溶液通过共缩聚合成技术

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

用于固相合成含有2&#39;-<em&gt; 0</em通过圆二色性修饰和表征

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

通过活性表面生长机制合成基片束缚金纳米线

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

直接溶液还原法快速合成金、钯、铂气凝胶的研究

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

乙腈绿色 Steglich 酯化合成酯的研究

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock copolymers are produced by one of three techniques: thin film rehydration, solvent switching or polymerization-induced self-assembly, and produce only flexible, polydisperse cylinders. Crystallization-driven self-assembly (CDSA) is a method which can produce cylinders with these properties, by stabilizing structures of a lower curvature due to the formation of a crystalline core. However, the living polymerization techniques by which most core-forming blocks are formed are not trivial processes and the CDSA process may yield unsatisfactory results if carried out incorrectly. Here, the synthesis of cylindrical nanoparticles from simple reagents is shown. The drying and purification of reagents prior to a ring-opening polymerization of &#949;-caprolactone catalyzed by diphenyl phosphate is described. This polymer is then chain extended by methyl methacrylate (MMA) followed by N,N-dimethyl acrylamide (DMA) using reversible addition&#8722;fragmentation chain-transfer (RAFT) polymerization, affording a triblock copolymer that can undergo CDSA in ethanol. The living CDSA process is outlined, the results of which yield cylindrical nanoparticles up to 500 nm in length and a length dispersity as low as 1.05. It is anticipated that these protocols will allow others to produce cylindrical nanostructures and elevate the field of CDSA in the future.

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of &#949;-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

通过结晶驱动生物降解块共聚物的自组装合成单分散圆柱状纳米粒子

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...