Name: expression and purification of nuclease-free oxygen scavenger protocatechuate 3,4-dioxygenase
Uploaded: 2019-11-08T13:00:00
Description: Watch this Scientific Journal Video about Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase at JoVE.com

这项工作得到了NIH GM121284和AI126742对KEY的支持。

1. 诱导大肠杆菌中的多氯二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二
<ol><li>在管中结合1 μL pVP91A-pcaHG多氯二苯并对二醇表达质粒（20纳克/μL，图1A）和20 μL大肠杆菌BL21（20 μL市售细胞，>2 x 10 6 cfu/μg质粒）。轻拂管子进行混合。将管子放在冰上 5 分钟。</li>
	<li>将变换置于 42°C 30 s。然后冰2分钟。</li>
	<li>添...

<ol>
	<li>Shera, E. B., Seitzinger, N. K., Davis, L. M., Keller, R. A., Soper, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+single+fluorescent+molecules.">Detection of single fluorescent molecules.</a> Chemical Physics Letters. 174 (6), 553-557 (1990).</li><li>Zheng, Q., Jockusch, S., Zhou, Z., Blanchard, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+reactive+oxygen+species+to+the+photobleaching+of+organic+fluorophores.">The contribution of reactive oxygen species to the photobleaching of organic fluorophores.</a> Photochemistry and Photobiology. 90 (2), 448-454 (2014).</li><li>Ha, T., Tinnefeld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photophysics+of+fluorescent+probes+for+single-molecule+biophysics+and+super-resolution+imaging.">Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging.</a> Annual Review of Physical Chemistry. 63, 595-617 (2012).</li><li>Dixit, R., Cyr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+damage+and+reactive+oxygen+species+production+induced+by+fluorescence+microscopy:+effect+on+mitosis+and+guidelines+for+non-invasive+fluorescence+microscopy.">Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy.</a> The Plant Journal: for Cell and Molecular Biology. 36 (2), 280-290 (2003).</li><li>Davies, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+species+formed+on+proteins+exposed+to+singlet+oxygen.">Reactive species formed on proteins exposed to singlet oxygen.</a> Photochemical &#38; Photobiological Sciences. 3 (1), 17-25 (2004).</li><li>Sies, H., Menck, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Singlet+oxygen+induced+DNA+damage.">Singlet oxygen induced DNA damage.</a> Mutation Research. 275 (3-6), 367-375 (1992).</li><li>Aitken, C. E., Marshall, R. A., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oxygen+scavenging+system+for+improvement+of+dye+stability+in+single-molecule+fluorescence+experiments.">An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments.</a> Biophysical Journal. 94 (5), 1826-1835 (2008).</li><li>Harada, Y., Sakurada, K., Aoki, T., Thomas, D. D., Yanagida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanochemical+coupling+in+actomyosin+energy+transduction+studied+by+in+vitro+movement+assay.">Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay.</a> Journal of Molecular Biology. 216 (1), 49-68 (1990).</li><li>Shi, X., Lim, J., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidification+of+the+oxygen+scavenging+system+in+single-molecule+fluorescence+studies:+in+situ+sensing+with+a+ratiometric+dual-emission+probe.">Acidification of the oxygen scavenging system in single-molecule fluorescence studies: in situ sensing with a ratiometric dual-emission probe.</a> Analytical Chemistry. 82 (14), 6132-6138 (2010).</li><li>Brown, C. K., Vetting, M. W., Earhart, C. A., Ohlendorf, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+analyses+of+designed+and+selected+mutants+of+protocatechuate+3,4-dioxygenase1.">Biophysical analyses of designed and selected mutants of protocatechuate 3,4-dioxygenase1.</a> Annual Review of Microbiology. 58, 555-585 (2004).</li><li>Senavirathne, G., 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=Widespread+nuclease+contamination+in+commonly+used+oxygen-scavenging+systems.">Widespread nuclease contamination in commonly used oxygen-scavenging systems.</a> Nature Methods. 12 (10), 901-902 (2015).</li><li>Senavirathne, G., Lopez, M. A., Messer, R., Fishel, R., Yoder, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+nuclease-free+protocatechuate+3,4-dioxygenase+for+prolonged+single-molecule+fluorescence+imaging.">Expression and purification of nuclease-free protocatechuate 3,4-dioxygenase for prolonged single-molecule fluorescence imaging.</a> Analytical Biochemistry. 556, 78-84 (2018).</li><li>Jones, N. 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=Retroviral+intasomes+search+for+a+target+DNA+by+1D+diffusion+which+rarely+results+in+integration.">Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.</a> Nature Communications. 7, 11409 (2016).</li><li>Liu, J., 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=Cascading+MutS+and+MutL+sliding+clamps+control+DNA+diffusion+to+activate+mismatch+repair.">Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair.</a> Nature. 539 (7630), 583-587 (2016).</li></ol>

氧气清除系统通常包含在单分子荧光显微镜中，以减少光漂白3，7，8。这些显微镜技术通常用于观察核酸或蛋白质与核酸1、13、14的相互作用。使用核酸酶污染OSS可能导致虚假结果。
商业上可用的开放苷和多氯丁二苯并对二苯并对二苯?...

单分子（SM）生物物理学是一个迅速成长的领域，它改变了我们对生物现象的观察方式。这一领域具有独特的能力，将物理和化学的基本定律与生物学联系起来。荧光显微镜是一种生物物理方法，可以达到SM灵敏度。荧光用于检测生物分子，将它们与小的有机荧光道或量子点1联系起来。这些分子在光漂白不可逆转之前，被激光激发时可以发射光子。当荧光标签受到化学损伤，破坏其在所需波长2，3处激发的能力时，就会发生光漂白。水缓冲液中活性氧物种（ROS）的存在是光漂白2，4的主要原因。此外，ROS可以破坏生物分子，并导致在SM实验5，6的错误观测。为了防止氧化损伤，氧气清除系统（OSS）可以使用3，7，8。葡萄糖氧化酶/催化酶（GODCAT）系统在去除氧8方面是有效的，但它作为中间体产生潜在的破坏性过氧化物。这些可能损害SM研究中感兴趣的生物分子。
或者，原生化液3，4二氧酶（PCD）将有效地去除O2从水溶液使用其基质原酸（PCA）7，9。PCD是一种金属酶酶，使用非铁来协调PCA，并使用溶解的O2 10催化catechol环开口反应。这一步反应被证明是一个整体更好的OSS，以提高荧光酸稳定性在SM实验7。不幸的是，许多市售的OSS酶，包括多氯二苯并对苯二苯醚，含有污染的核酸酶11。这些污染物可能导致 SM 实验中使用的核酸基板损坏。这项工作将阐明在SM系统中使用重组PCD的基于色谱的纯化方案。PCD 可广泛应用于 ROS 破坏数据采集所需基板的任何实验。

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td>&#946;ME</td>
		</tr>
		<tr>
			<td>30% acrylamide and bis-acrylamide solution, 29:1</td>
			<td>Bio-Rad</td>
			<td>161-0156</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid, Glacial Certified ACS</td>
			<td>Fisherl Chemical</td>
			<td>A38C-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, Granulated</td>
			<td>BD Biosciences</td>
			<td>DF0145-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA FPLC System</td>
			<td>GE Healthcare Life Sciences</td>
			<td>AKTA Purifier: Box-900, pH/C-900, UV-900, P-900, and Frac-920</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit</td>
			<td>EMD Millipore</td>
			<td>UFC201024</td>
			<td>10 kDa MWCO</td>
		</tr>
		<tr>
			<td>Ammonium iron(II) sulfate hexahydrate</td>
			<td>Sigma</td>
			<td>F-2262</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate (APS) Tablets</td>
			<td>Amresco</td>
			<td>K833-100TABS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Amresco</td>
			<td>0339-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Tryptone</td>
			<td>BD Biosciences</td>
			<td>DF0123173</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Bacto Dehydrated Culture Media Additive: Bottle Yeast Extract</td>
			<td>VWR</td>
			<td>90004-092</td>
			<td></td>
		</tr>
		<tr>
			<td>BIS-TRIS propane,&#62;=99.0% (titration)</td>
			<td>Sigma-Aldrich</td>
			<td>B6755-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue</td>
			<td>Amresco</td>
			<td>0472-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 96&#8211;Well Flat&#8211;Bottom EIA Plate</td>
			<td>Bio-Rad</td>
			<td>2240096EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>P212121</td>
			<td>SV-DTT</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline 500ML</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1302</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G37-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated LB Broth Miller</td>
			<td>EMD Biosciences</td>
			<td>1.10285.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hi-Res Standard Agarose</td>
			<td>AGTC Bioproducts</td>
			<td>AG500D1</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I0250-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Goldbio</td>
			<td>I2481C25</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Roche</td>
			<td>11017128001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme from Chicken Egg White</td>
			<td>Sigma-Aldrich</td>
			<td>L6876-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>Amresco</td>
			<td>0288-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvolume Spectrophotometer, with cuvet capability</td>
			<td>Thermo Fisher</td>
			<td>ND-2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>P212121</td>
			<td>RP-S23020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow (100 ml)</td>
			<td>Qiagen</td>
			<td>30430</td>
			<td></td>
		</tr>
		<tr>
			<td>Novagen BL21 Competent Cells</td>
			<td>EMD Millipore</td>
			<td>69-449-3</td>
			<td>SOC media included</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Fisher Scientific</td>
			<td>0-267</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Gold Biotechnology</td>
			<td>P-020-25</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Amresco</td>
			<td>0754-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protocatechuic acid</td>
			<td>Fisher Scientific</td>
			<td>ICN15642110</td>
			<td>PCA</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>P212121</td>
			<td>CI-00270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M2 Microplate Reader</td>
			<td>Molecular Devises</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Filter Units with PES Membrane &#62; 250mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>09-741-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Filter Units with PES Membrane &#62; 500mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>09-741-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Superose 12 10/300 GL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17517301</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Amresco</td>
			<td>0761-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Ultra Pure</td>
			<td>Gojira Fine Chemicals</td>
			<td>UTS1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon 9410 variable mode fluorescent imager</td>
			<td>GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure EDTA</td>
			<td>Invitrogen/Gibco</td>
			<td>15575</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>208086</td>
			<td></td>
		</tr>
	</tbody>
</table>

expression and purification of nuclease-free oxygen scavenger protocatechuate 3,4-dioxygenase

单分子（SM）显微镜用于实时研究荧光体标记生物分子的动态分子相互作用。然而，荧光团容易通过溶解氧（O2）的光漂白失去信号。为了防止光漂白和延长荧光团的寿命，采用氧气清除系统（OSS）来减少O2。市售的OSS可能被破坏或降解核酸的核酸污染，混淆了实验结果的解释。在这里，我们详细介绍了高活性伪单核素的表达和纯化方案，即原氧化酶-3，4-二氧酶（PCD），没有可检测到的核酸酶污染。PCD通过将基质原酸（PCA）转化为3-卡博西-cis-muconic酸，可以有效地去除活性O2种。此方法可用于 O2在数据采集中起到不利作用的任何水性系统。与市售的多氯二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二者相比，该方法能有效地产生高活性、无核酸酶的PCD。

市售的氧气清除剂PCD经常受到DNA核酸酶的污染。核酸酶活性的污染可能导致荧光研究的虚假结果，特别是分析DNA或DNA相互作用蛋白质的研究。我们发现，重组PCD，六聚二醇标记pcaH和pcaG的异体，可以表达在大肠杆菌（图1）。异质器首先通过镍亲和色谱法进行纯化（图2）。PCD在二分之二浓度的2个步骤中洗脱。色谱分数由SDS-PAGE分...

Watch this Scientific Journal Video about Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase at JoVE.com

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

原酸3，4-二氧酶（PCD）可以酶分从水系统中分离游离的游离双原子氧，使用其基质原酸（PCA）。该协议描述了这种氧气清除酶的表达、纯化和活性分析。

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

我们的协议产生高活性PCD，不含可检测的核酸酶，可用于任何水氧是污染物的解决方案。我们使用的技术易于复制，并确保产品中不存在核酸酶污染物。首先，将一个微升的 pVP91A-pcaHG PCD 表达质粒和 20 微升的商用大肠杆菌 BL21 组合在一个管子中。轻拂管子混合，将管子放在冰上五分钟。为了继续转化反应，将管子在42摄氏度的水浴中放置30秒，然后在冰上放置两分钟。加入 80 微升 SOC 介质，在 225 rpm 和 37 摄氏度下摇动一小时。接下来，将转化反应混合物倒入LB-安西林加糖上，并使用板扩散器将混合物扩散。用盖子盖住盘子，在37摄氏度下孵育板16至18小时。之后，从盘子中挑选一个菌落，在250毫升的埃伦迈尔烧瓶中接种50微升的LB-安皮西林。以 225 rpm 的速度将烧瓶放在摇床上，在 37 摄氏度下孵育 16 至 18 小时。然后，将20毫升的孵育培养基转移到一个四升烧瓶中，里面装满了一升LB-amp机素。在 37 摄氏度下以 225 rpm 转速摇动。每小时，在光度计上将一毫升培养力绘制成一个培养盒，以600纳米的速度测量光学密度。当培养光密度增加近 0.5 时，将测量频率增加至每 15 分钟一次，直到 OD600 达到 0.5。然后，将四升烧瓶转移到冰箱中。在冰浴中旋转烧瓶以降低培养温度。在 17 摄氏度和 180 rpm 的温度下将四升烧瓶转移到孵化器中。继续每 20 分钟监控一次 OD600。在0.7 OD600时，加入IPTG和硫酸铵六氢水合物溶液，最终浓度分别达到0.5毫摩尔和10毫克/升。在 180 rpm 和 17 摄氏度下摇动培养力 18 小时。孵育后，将培养瓶放在冰中两分钟。在1.5毫升聚丙烯微离心管中收获一毫升诱导细胞，用于SDS-PAGE分析。接下来，将细菌培养剂倒入瓶子中，每瓶500毫升，进行离心。在摄氏4度和3000次g下培养20分钟。去位超级纳特人。移液器以重新分配颗粒，每粒在12.5毫升的冷PBS中，并在50毫升锥形管中每两个再增射。再次像以前一样将单元格取出。然后，丢弃超级纳特，并使用移液器在每个管中加入10毫升的解液缓冲液，以重新暂停细胞。用诱导细胞对管子进行声波化后，将细菌酸盐倒入预冷的聚碳酸酯瓶中。在12万次g和4摄氏度的温度下离心60分钟，将上清液倒入一个冷的50毫升锥形管中。使用镀镍树脂柱制备 FPLC 仪器后，以每分钟 0.15 毫升的速度将样品加载到柱上。用20毫升镍缓冲液用20毫升的镍缓冲液清洗柱。将洗涤液保留在 50 毫升管中进行分析。接下来，用15毫升镍缓冲液用125毫升的二米多摩尔洗涤柱。收集洗脱在19个分数0.8毫升每个。再次用 15 毫升 100%镍缓冲液 B 洗涤柱，并额外收集 75 分，每个 0.2 毫升。继续分析 12%SDS-PAGE 凝胶上收集的分数，以确认 PCD 的存在。首先，在1.5毫升聚丙烯微离心管中加入35微升反应缓冲液。在管中，结合峰色谱分数的五微升和 500 纳米的三千基对超焦质质 pXba。将管子放在37摄氏度的水浴中一小时。根据手稿制备阿加罗斯凝胶后，在凝胶中加入每种反应的30微升。在环境温度下，电泳在 10 伏/厘米下约 1 小时。现在，橙色的G染料已经到达凝胶的末尾。立即使用荧光扫描仪成像凝胶的溴化乙基信号。利用强度和图像像素大小的功能，确定各种DNA物种的像素体积，如超高、线性和刻痕圆。要确定每个DNA物种的百分比，请将单个DNA物种的像素体积除以所有单个物种的总像素体积。SDS-PAGE 分析表明，第二个洗脱峰含有最小的蛋白质污染，这表示几乎纯PCD异质剂和最小到检测不到的核酸酶活性。因此，将这两个PCD洗脱的10个分数组合在一个五毫升的圆锥管中，用于进一步分析。PCD大小排除色谱纯化后，在4摄氏度的冷室中，将微离心管中适当体积的氯化钠、盐酸三氧化三、氯化镁、二硫化物、PCA和超冷质粒pXba组合成5X库存。将 96 孔平底板放在冰上，将 10 微升组合的 5X 溶液转移到油井中。在分析之前，立即向每个井中加入 10 微升的单个 PCD SEC 分数。将板读卡器设置为 37 摄氏度的内部温度，然后将 96 井板转移到板架上。将板架缩回仪器中，以 20 秒的间隔以 290 纳米为单位测量吸光度一小时。将仪器设置为每次读数前五秒钟摇动板。一小时后，向每井添加10微升停止溶液以终止反应。对 PCD SEC 分数执行凝胶电泳。选择具有最多 PCA 氧化活性的分数，如吸收率降低和未观察到的核酸酶污染。测量280纳米的吸光度，根据吸光度和消光系数计算PCD总浓度。将所选分数存放在零下 80 摄氏度下进行长期存储。在这个实验中，发现重组PCD，一种六氯苯二丁标记pcaH和pcaG的异质体，用大肠杆菌表达。异质首先通过镍亲和力色谱进行纯化。吸收度显示为蓝色，镍缓冲液 B 的浓度百分比显示为红色。流经显示未与镍树脂结合的可溶性细菌蛋白。一些PCD在125毫摩尔伊米达佐尔的存在下洗清，但大多数蛋白质在250毫摩尔伊米达佐尔中洗清。代表性 SDS-PAGE 分析表明，洗脱步骤中的分数没有污染物。核酸酶测定的阿加罗斯凝胶通过比较与没有添加蛋白质的负对比和被DNA核酸酶污染的阳性对照物来揭示污染。在阿加罗斯凝胶核酸酶测定中观察到的各种DNA物种的定量显示，29至38的片段几乎没有核酸酶活性，被SEC选择进行组合、浓缩和进一步纯化。按照此过程，纯化的 PCD 可以在所需的氧气清除系统中使用和优化。这将确定系统所需的 PCD 的确切数量。在单分子显微镜实验中，该技术已用于延长荧光寿命，从而可以延长数据收集时间。使用的超离心量是危险的。在旋转之前，确保离心机保持适当平衡。溴化钛是一种危险的试剂。穿戴个人防护装备避免接触，并妥善处置。

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Expression, Purification, and Antimicrobial Activity of S100A12

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Expression and Purification of Mammalian Bestrophin Ion Channels

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

用于稳定碳氧同位素分析的牙釉质碳酸盐的取样与预处理

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

真核硼酸背液转运体的表达、增溶及纯化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...