Name: formulation and characterization of bioactive agent containing nanodisks
Uploaded: 2023-03-17T14:15:00
Description: Watch this Scientific Journal Video about Formulation and Characterization of Bioactive Agent Containing Nanodisks at JoVE.com

这项工作得到了美国国立卫生研究院（R37 HL-64159）的资助。

1. 支架蛋白组分的转化、表达和纯化
<ol><li>BL21 细菌转化与含 apoE4-NT 的质粒<ol><li>将一管BL21（DE3）感受态细胞在冰上解冻10分钟。</li>
		<li>所有冰融化后，轻轻混合并小心地将 50 μL 细胞移液到冰上的转化管中。</li>
		<li>向细胞混合物中加入含有 50 ng 质粒 DNA（有关序列，参见 补充表 1）的 5 μL。小心地轻弹试管四到五次以混合。不要涡旋。</li>
		<li>将混合物放在冰上30?...

<ol>
	<li>Fox, C. A., Moschetti, A., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+HDL+as+a+therapeutic+delivery+device.">Reconstituted HDL as a therapeutic delivery device.</a> Biochimica et Biophysica Acta. Molecular and Cell Biology of Lipids. 1866 (11), 159025 (2021).</li><li>Ong, K. L., Cochran, B. J., Manandhar, B., Thomas, S., Rye, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL+maturation+and+remodelling.">HDL maturation and remodelling.</a> Biochimica et Biophysica Acta. Molecular and Cell Biology of Lipids. 1867 (4), 159119 (2022).</li><li>Nicholls, S. 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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodisks+derived+from+amphotericin+B+lipid+complex.">Nanodisks derived from amphotericin B lipid complex.</a> Journal of Pharmaceutical Sciences. 97 (10), 4425-4432 (2008).</li><li>Nguyen, T. S., 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=Amphotericin+B+induces+interdigitation+of+apolipoprotein+stabilized+nanodisk+bilayers.">Amphotericin B induces interdigitation of apolipoprotein stabilized nanodisk bilayers.</a> Biochimica et Biophysica Acta. 1778 (1), 303-312 (2008).</li><li>Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobiotechnology+applications+of+reconstituted+high+density+lipoprotein.">Nanobiotechnology applications of reconstituted high density lipoprotein.</a> Journal of Nanobiotechnology. 8, 28 (2010).</li><li>Lalefar, N. R., Witkowski, A., Simonsen, J. B., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt3a+nanodisks+promote+ex+vivo+expansion+of+hematopoietic+stem+and+progenitor+cells.">Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells.</a> Journal of Nanobiotechnology. 14 (1), 66 (2016).</li><li>Crosby, N. M., 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=Anti-CD20+single+chain+variable+antibody+fragment-apolipoprotein+A-I+chimera+containing+nanodisks+promote+targeted+bioactive+agent+delivery+to+CD20-positive+lymphomas.">Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.</a> Biochemistry and Cell Biology. 93 (4), 343-350 (2015).</li><li>Ghosh, M., Ren, G., Simonsen, J. B., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid+nanodisks+as+an+siRNA+delivery+vehicle.">Cationic lipid nanodisks as an siRNA delivery vehicle.</a> Biochemistry and Cell Biology. 92 (3), 200-205 (2014).</li><li>Fox, C. A., Ellison, P., Ikon, N., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-induced+transformation+of+cardiolipin+nanodisks.">Calcium-induced transformation of cardiolipin nanodisks.</a> Biochimica et Biophysica Acta. Biomembranes. 1861 (5), 1030-1036 (2019).</li><li>Fox, C. A., Lethcoe, K., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-induced+release+of+cytochrome+c+from+cardiolipin+nanodisks:+Implications+for+apoptosis.">Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis.</a> Biochimica et Biophysica Acta Biomembranes. 1861 (12), 183722 (2021).</li><li>Fox, C. A., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+binding+assay+reveals+doxorubicin+preference+for+DNA+versus+cardiolipin.">Dye binding assay reveals doxorubicin preference for DNA versus cardiolipin.</a> Analytical Biochemistry. 594, 113617 (2020).</li><li>Fox, C. A., Romenskaia, I., Dagda, R. K., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+nanodisks+confer+protection+against+doxorubicin-induced+mitochondrial+dysfunction.">Cardiolipin nanodisks confer protection against doxorubicin-induced mitochondrial dysfunction.</a> Biochimica et Biophysica Acta Biomembranes. 1864 (10), 183984 (2022).</li></ol>

含有纳米盘的生物活性剂的配制提供了一种溶解否则不溶的疏水化合物的便捷方法。由于产品生物活性剂纳米盘完全溶于水介质，因此它们为各种疏水分子提供了一种有用的递送方法（表1）。这些包括小分子、天然和合成药物、植物营养素、激素等。配方策略通常遵循标准方案，该方案必须考虑生物活性剂在有机溶剂中的溶解度特性。除了选择合适的有机溶剂来溶解生物活性剂外，?...

新生的盘状高密度脂蛋白（HDL）是人体循环系统中更丰富的球形高密度脂蛋白的天然祖先。这些新生颗粒，也称为pre-ß HDL，具有独特而独特的结构特性1。事实上，新生的HDL不是作为球状粒子存在的，而是圆盘状的。对天然和重组盘状高密度脂蛋白的广泛结构表征研究表明，它们由磷脂双层组成，其周长由两亲性可交换载脂蛋白（apo）限制，例如apoA-I。在人脂蛋白代谢中，循环的新生高密度脂蛋白从外周细胞中积累脂质，并在依赖于关键蛋白质介质的过程中成熟为球形高密度脂蛋白，包括ATP结合盒转运蛋白A1和卵磷脂：胆固醇酰基转移酶2。这个过程代表了反向胆固醇转运途径的关键组成部分，被认为可以预防心脏病。有了这些知识和重建盘状HDL的能力，研究人员将这些颗粒用作治疗动脉粥样硬化的治疗干预措施3。从本质上讲，将重组高密度脂蛋白（rHDL）输注到患者中会促进胆固醇从斑块沉积物中排出，并将其返回到肝脏以转化为胆汁酸并从体内排泄。一些生物技术/制药公司正在推行这种治疗策略4。
与此同时，在实验室中产生这些粒子的能力引发了一系列研究活动，导致了新的应用和新技术。一个突出的应用涉及使用rHDL颗粒作为微型膜，在天然环境中容纳跨膜蛋白5。迄今为止，已有数百种蛋白质成功掺入盘状rHDL，研究表明，这些蛋白质作为受体、酶、转运蛋白等保留了天然构象和生物活性。这些被称为“纳米盘”的颗粒也被证明适合结构表征，通常以高分辨率6。这种跨膜蛋白研究方法被认为优于洗涤剂胶束或脂质体的研究，因此正在迅速发展。重要的是要认识到，已经报道了两种能够形成rHDL的不同方法。“胆酸盐透析”方法13在与在rHDL双层5中掺入跨膜蛋白相关的应用中很常用。基本上，该配制方法涉及在含有洗涤剂胆酸钠（或脱氧胆酸钠;胶束分子量[MW]为4，200Da）的缓冲液中混合形成磷脂的双层，支架蛋白和目标跨膜蛋白。去垢剂可有效溶解不同的反应组分，使样品能够针对缺乏去垢剂的缓冲液进行透析。在透析步骤中，当洗涤剂从样品中取出时，会自发形成rHDL。当这种方法用于捕获感兴趣的跨膜蛋白时，产物颗粒被称为纳米盘5。然而，尝试使用这种方法掺入小分子疏水生物活性剂（MW <1，000 Da）在很大程度上是不成功的。与跨膜蛋白不同，小分子生物活性剂能够与洗涤剂一起从透析袋中逸出，大大降低了它们在rHDL中的掺入效率。通过从配方混合物14中省略洗涤剂解决了这个问题。相反，组分依次加入到水性缓冲液中，从双层形成脂质开始，形成含有rHDL的稳定生物活性剂，称为纳米盘。其他人则使用rHDL掺入和运输体内成像剂7。最近，由载脂蛋白支架和阴离子甘油磷脂心磷脂组成的专用rHDL已被用于配体结合研究。这些颗粒为研究心磷脂与各种水溶性配体（包括钙、细胞色素c和抗癌剂阿霉素8）的相互作用提供了一个平台。
本研究的重点是配制具有稳定掺入疏水性生物活性剂（即纳米盘）的rHDL。这些试剂整合到盘状rHDL颗粒的脂质环境中的能力有效地赋予它们水溶性。因此，纳米盘具有 体内 治疗应用的潜力。在配制纳米盘时，需要特定的孵育/反应条件才能成功地将离散疏水性生物活性剂掺入产品颗粒中，本报告的目的是提供详细的实用信息，可用作为特定应用创建新型纳米盘颗粒的基础模板。因此，在本手稿的上下文中，术语nanodisc和nanodisk是不可互换的。纳米盘是指配制为含有嵌入其脂质双层5中的跨膜蛋白的rHDL，而术语纳米盘是指配制为掺入低分子量（<1，000 Da）疏水性生物活性剂（例如两性霉素B14）的rHDL。
有多种方法可用于获取合适的支架蛋白。可以从制造商处购买支架蛋白[例如apoA-I（SRP4693）或apoE4（A3234）]，但是，成本可能是一个限制因素。首选方法是在大肠杆菌中表达重组支架蛋白。针对人类apoA-I9，apoE410以及昆虫血淋巴蛋白apolipophorin-III11的协议已发布。为了本文描述的实验的目的，使用重组人apoE4的N端（NT）结构域（氨基酸1-183）。合成编码人apoE4-NT的核苷酸序列并将其插入到与载体编码的pelB前导序列直接相邻的pET-22b（+）表达载体中。该构建体导致pelB领导序列-apoE4-NT融合蛋白的表达。蛋白质合成后，细菌pelB领导序列将新合成的蛋白质引导到周质空间，其中前导肽酶切割pelB序列。所得的apoE4-NT蛋白没有序列标签或尾巴，随后逃逸细菌并积聚在培养基11，12中，简化了下游加工。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>Cayman Chemical Company</td>
			<td>11636</td>
			<td>ND Formulation &#38; Standard Preparation</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP17925</td>
			<td>Transformation &#38; Expansion</td>
		</tr>
		<tr>
			<td>ApoE4-NT Plasmid</td>
			<td>GenScript</td>
			<td>N/A</td>
			<td>Transformation</td>
		</tr>
		<tr>
			<td>Baffled Flask</td>
			<td>New Brunswick Scientific</td>
			<td>N/A</td>
			<td>Expansion &#38; Expression</td>
		</tr>
		<tr>
			<td>BL21 competent E coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td>Transformation</td>
		</tr>
		<tr>
			<td>Centrifuge bottles</td>
			<td>Nalgene</td>
			<td>3140-0250</td>
			<td>Expression</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>G607-4</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>472301</td>
			<td>Standard Prepartation</td>
		</tr>
		<tr>
			<td>Dymyristoylphosphatidylcholine</td>
			<td>Avanti Lipids</td>
			<td>850345P</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>Erlenmeyer flask</td>
			<td>Bellco Biotechnology</td>
			<td>N/A</td>
			<td>Expansion &#38; Expression</td>
		</tr>
		<tr>
			<td>Falcon Tubes</td>
			<td>Sarstedt Ag &#38; Co</td>
			<td>D51588</td>
			<td>Yeast Viability Assay</td>
		</tr>
		<tr>
			<td>Glass borosilicate tubes</td>
			<td>VWR</td>
			<td>47729-570</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>GraphPad (Software)</td>
			<td>Dotmatics</td>
			<td>N/A</td>
			<td>Yeast Viability Assay</td>
		</tr>
		<tr>
			<td>Heated Sonication Bath</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>ND Formulaton</td>
		</tr>
		<tr>
			<td>Heating and Nitrogen module</td>
			<td>Thermo Scientific</td>
			<td>TS-18822</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>HiTrap Heparin HP (5 mL)</td>
			<td>GE Healthcare</td>
			<td>17-0407-03</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP1755</td>
			<td>Expression</td>
		</tr>
		<tr>
			<td>J-25 Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>J325-IM-2</td>
			<td>Expression</td>
		</tr>
		<tr>
			<td>JA-14 Rotor</td>
			<td>Beckman Coulter</td>
			<td>339247</td>
			<td>Expression</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7755030</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>Praxair</td>
			<td>UN1066</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>NZCYM media</td>
			<td>RPI Research Products</td>
			<td>N7200-1000.0</td>
			<td>Expansion &#38; Expression</td>
		</tr>
		<tr>
			<td>Pet-22B vector</td>
			<td>GenScript</td>
			<td>N/A</td>
			<td>Transformation</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875718</td>
			<td>Transformation &#38; Expansion</td>
		</tr>
		<tr>
			<td>Quartz Cuvettes</td>
			<td>Fisher Brand</td>
			<td>14385 928A</td>
			<td>Spectral Analysis</td>
		</tr>
		<tr>
			<td>Shaking Incubator</td>
			<td>New Brunswick Scientific</td>
			<td>M1344-0004</td>
			<td>Transformation, Expansion, &#38; Expression</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer Buoys</td>
			<td>Thermo Scientific</td>
			<td>66430</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing</td>
			<td>Thermo Scientific</td>
			<td>88243</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Sodium Phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP329-500</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Spectra/POR Weighted Closures</td>
			<td>Spectrum Medical Industries</td>
			<td>132736</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Shimadzu UV-1800</td>
			<td>220-92961-01</td>
			<td>spectral analysis</td>
		</tr>
		<tr>
			<td>Tabletop Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>366816</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>UVProbe 2.61 (Software)</td>
			<td>Shimadzu</td>
			<td>N/A</td>
			<td>Spectral Analysis</td>
		</tr>
		<tr>
			<td>Vacuum filter</td>
			<td>Millipore</td>
			<td>9004-70-0</td>
			<td>Expression &#38; Purification</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>GAST Manufacturing Inc</td>
			<td>DOA-P704-AA</td>
			<td>Expression &#38; Purification</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>12-812</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td>N/A</td>
			<td>BY4741</td>
			<td>Yeast Viability Assay</td>
		</tr>
		<tr>
			<td>Yeast Extract-Peptone-Dextrose</td>
			<td>BD</td>
			<td>242820</td>
			<td>Yeast Viability Assay</td>
		</tr>
	</tbody>
</table>

formulation and characterization of bioactive agent containing nanodisks

术语纳米盘是指由双层形成脂质、支架蛋白和集成生物活性剂组成的离散类型的纳米颗粒。纳米盘被组织成圆盘状的脂质双层，其周边由支架蛋白限制，支架蛋白通常是可交换载脂蛋白家族的成员。许多疏水性生物活性剂通过集成到颗粒脂质双层的疏水环境中，在纳米盘中有效地溶解，产生了直径为10-20nm的颗粒群。纳米盘的配制需要单个组分的精确比例，每种组分的适当顺序添加，然后对配方混合物进行浴超声处理。两亲性支架蛋白自发接触并重组分散的双层形成脂质/生物活性剂混合物，形成离散、均匀的纳米盘颗粒群。在此过程中，反应混合物从不透明、浑浊的外观转变为澄清的样品，当完全优化时，离心时不会产生沉淀。表征研究涉及生物活性剂增溶效率的测定、电子显微镜、凝胶过滤色谱、紫外可见（UV/Vis）吸光光谱和/或荧光光谱。随后通常使用培养的细胞或小鼠研究生物活性。在含有抗生素（即大环内酯类多烯类抗生素两性霉素B）的纳米盘的情况下，可以测量它们作为浓度或时间函数抑制酵母或真菌生长的能力。相对容易配制、相对于组分的多功能性、纳米级粒径、固有稳定性和水溶性允许纳米盘技术的无数体 外 和 体内 应用。在本文中，我们描述了一种配制和表征含有两性霉素B作为疏水生物活性剂的纳米盘的一般方法。

生物活性剂纳米盘配制工艺 在所描述的ampB-纳米盘配制程序中，当样品外观从浑浊转变为透明时，反应被认为是完成的（图1）。这种变化表明纳米盘已经形成并且生物活性剂已经溶解。通常，生物活性剂吸收可见波长区域的光（例如，ampB，姜黄素，叶黄素，辅酶Q10），在这些情况下，样品采用生物活性剂的颜色。样品澄清完成后（通常为浴超声处理5...

Watch this Scientific Journal Video about Formulation and Characterization of Bioactive Agent Containing Nanodisks at JoVE.com

Formulation and Characterization of Bioactive Agent Containing Nanodisks

在这里，我们描述了含有纳米盘的生物活性剂的生产和表征。以两性霉素B纳米盘为例，逐步描述该方案。

含纳米片生物活性剂的配制与表征

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

该协议提供了实用知识，可用于配制纳米级颗粒，即纳米盘，其稳定地掺入广泛的疏水性生物活性分子。这种方法有许多实际应用。赋予其他不溶性生物活性化合物水溶性的能力允许在药物输送应用中使用纳米盘。所描述的方法很简单。优选在所描述的规模上进行研究，五毫克磷脂，两毫克支架蛋白，以便通过目视检查容易地监测纳米盘的形成。展示该程序的将是Ryan实验室的本科生研究员Michael Karo和Matthew Swackhammer。要制备磷脂等分试样，请称取五毫克DMPC并将其转移到玻璃试管中。通过加入300微升氯仿和100微升甲醇溶解磷脂，比例为三比一。通过将玻璃试管置于温和的氮气流下10至15分钟来蒸发有机溶剂，使得沿管底部壁形成一层干燥的磷脂薄膜。为了配制两性霉素B或AmpB-纳米盘，将450微升PBS移液到冻干的磷脂等分试样中，并涡旋30秒以分散脂质。将50微升20毫克每毫升储备AmpB溶液移液到分散的磷脂样品中并涡旋。向含有分散磷脂和AmpB的玻璃试管中加入500微升每毫升4毫克的ApoE4 NT支架蛋白。浴在24摄氏度下超声处理样品，直到溶液澄清。对于AmpB-纳米盘透析，准备一段透析管，将其彻底浸泡在蒸馏去离子水中10分钟。使用透析管夹，夹住浸泡透析管的一端，确保没有液体逸出。将一个狭窄的颈部漏斗插入透析管的开口端，并将 AmpB-纳米盘样品转移到透析管中。拆下漏斗，并用另一个透析管夹夹住透析管的末端。将泡沫透析浮子放在透析管的密封端之一上，然后将组装好的透析管放入装有新鲜制备的一升PBS缓冲液的烧杯中。将磁力搅拌棒放入烧杯底部，并将搅拌控制调低，确保不会产生涡流。让透析在四摄氏度下持续过夜。通过翻转电源开关打开分光光度计，并通过按PC控制连接到相应的支持计算机。在支持计算机上，打开标有UVProbe 2.61的软件，然后单击连接到分光光度计“连接”在左下角。单击频谱，然后单击方法“在顶部工具栏中。单击测量选项卡，然后在位于波长范围内的开始“文本框中输入 500，在结束”文本框中输入 300。单击标有“扫描速度”的选项卡旁边的下拉菜单，并将其设置为中。通过将一毫升DMSO转移到两个石英比色皿中来制备样品空白。将两个比色皿装入分光光度计的相应样品端口。点击 自动空白“并通过单击记录从 300 到 500 纳米的光谱 Start 开始”在左下角。对于 AmpB 标准光谱分析，从前样品口取出比色皿，每毫升 AmpB 储备溶液加入 20 微升 1 毫克。将比色皿装入样品板，然后单击开始“以记录样品吸光度。记录样品吸光度后，取出比色皿并将液体内容物倒入废物容器中。用去离子水洗三次彻底冲洗比色皿，然后用70%乙醇洗涤三次。对于破碎的AmpB-纳米盘的光谱分析，通过将每毫升AmpB-Nanodisk原液中的20微升1毫克移液到1毫升DMSO中来制备样品，并在记录光谱之前孵育一分钟。将比色皿装入前部样品端口，然后单击开始“以记录样品吸光度。对于PBS缓冲液空白的光谱分析，通过将一毫升PBS转移到两个石英比色皿中来制备空白。将比色皿装入样品端口。单击自动空白“并记录从300到500纳米的光谱。对于未中断的AmpB-纳米盘样品的光谱分析，从前样品端口取出比色皿，并将20微升每毫升1毫克AmpB-纳米盘样品添加到PBS缓冲液中。将比色皿装回样品板。单击开始“并记录样品光谱。当样品外观从浑浊转变为透明时，AmpB-纳米盘配方反应被认为是完成的。显示了DMSO和PBS中AmpB样品的紫外可见吸收光谱。在DMSO中，在372、392和415纳米处观察到三个不同的吸光度最大值，即指示AmpB的峰。PBS中AmpB-纳米盘的吸光度光谱在较短波长处显示出单个主吸光峰。相比之下，DMSO中AmpB纳米盘的吸光度光谱与储备AmpB的光谱相似。AmpB-纳米盘的生物活性是通过评估酵母生长抑制测定来进行的。PBS、DMSO和重组高密度脂蛋白等对照样品表明，AmpB以外的纳米盘成分对酵母生长没有明显影响。阳性对照证实AmpB是酵母生长的有效抑制剂。在AmpB-纳米盘的情况下，观察到浓度依赖性生长抑制活性。并非所有的纳米磁盘制备都是一样的。有些可能需要更多时间、不同的脂质或不同的支架蛋白。它们只是模糊不清}是纳米盘样品的澄清。该技术导致了用于研究阿霉素诱导的心脏毒性、细胞凋亡、配体相互作用的新型纳米颗粒，并递送了许多水性不溶性生物活性分子，包括叶黄素和姜黄素。

Research

JoVE Journal

Biochemistry

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

结合干湿实验室技术指导大型卷曲螺旋含有蛋白质的结晶

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

科研

生物化学

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

植物甘露聚糖细胞壁多糖的结构表征

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterases 在抗杀虫剂家蝇、家蝇中的功能表征

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

生物大分子晶体的完全自主表征及数据采集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

使用含有微型基因的Alu元素来分析圆形RNA

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...