Name: efficient purification and lc-ms/ms-based assay development for ten-eleven translocation-2 5-methylcytosine dioxygenase
Uploaded: 2018-10-15T13:00:00
Description: Watch this Scientific Journal Video about Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase at JoVE.com

这项研究由美国国防部资助的形式的想法奖 (W81XWH-13-1-0174), 再生障碍性贫血 & MDS 基金会赠款, 和 UMRB 授予 M.M. 作者感谢莫希特贾斯瓦尔和 Subhradeep 巴尔在 TET2 向量的初始克隆 pDEST14。

1. 无标记人 TET2 酶的克隆和纯化
<ol><li>使用特定于站点的重组技术将人类 TET2 酶 (TET2 1129-1936、Δ1481-1843) 克隆到 pDEST14 目标向量中, 如前所述22。 注意: 以前的研究表明, C 终端 TET2 酶 (TET2 1129-1936, Δ1481-1843) 域是最小催化活动域21,23。为了用 pDONR221 向量表达未标记的 TET2 酶域, 在 PCR 过程中将发光达尔加诺和科扎克序列合并到正向底漆...

<ol>
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F., 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=Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA.">Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.</a> Science. 333, 1303-1307 (2011).</li><li>Ponnaluri, V. K., Maciejewski, J. P., Mukherji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanistic+overview+of+TET-mediated+5-methylcytosine+oxidation.">A mechanistic overview of TET-mediated 5-methylcytosine oxidation.</a> Biochemical and biophysical research communications. 436, 115-120 (2013).</li><li>Tamanaha, E., Guan, S., Marks, K., Saleh, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributive+Processing+by+the+Iron(II)/alpha-Ketoglutarate-Dependent+Catalytic+Domains+of+the+TET+Enzymes+Is+Consistent+with+Epigenetic+Roles+for+Oxidized+5-Methylcytosine+Bases.">Distributive Processing by the Iron(II)/alpha-Ketoglutarate-Dependent Catalytic Domains of the TET Enzymes Is Consistent with Epigenetic Roles for Oxidized 5-Methylcytosine Bases.</a> Journal of the American Chemical Society. 138, 9345-9348 (2016).</li><li>Ko, 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=Impaired+hydroxylation+of+5-methylcytosine+in+myeloid+cancers+with+mutant+TET2.">Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.</a> Nature. 468, 839-843 (2010).</li><li>Langemeijer, S. 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TET2 基因突变是在不同的造血恶性肿瘤患者中发现的最常见的遗传变化。到目前为止, 数以百计的不同的 TET2 突变, 其中包括废话, 帧移位, 和错义突变, 已确定在患者12。与 wt-TET214相比, TET2突变的患者在骨髓中的基因组5hmC 水平低。突变TET2实验概括了这些突变对转染细胞中5hmC 水平的影响14。结果 TET2-knockout 小鼠模型表明, TET2 酶水?...

在 CpG 二核苷酸内胞嘧啶碱基的 C5 位置是哺乳动物基因组1中主要的甲基化部位 (5mCpG)。此外, 一些最近的研究发现在非 CpG 站点 (5mCpH, 其中 H = a, T, 或 C)2,3的广泛 C5 胞嘧啶甲基化 (5mC)。5mC 修饰在内源座子和基因促进剂3、4、5中用作转录消声器。5mC DNA 甲基化在 X 染色体失活、基因印迹、核重编程和组织特异基因表达5、6、7等方面也起着重要作用。C5 位置的胞嘧啶甲基化是由 DNA 甲基转移酶进行的, 而这些酶的突变导致重大发育缺陷8。5mC 标记的去除由 TET1-3 5mC 氧化酶9、10启动。这些春节家庭芳香烃通过连续氧化步骤11,12,13转换5mC 到 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) 和 5-carboxylcytosine (5caC)。最后, 胸腺嘧啶 DNA glycosylase 替代5fC 或5caC 到未修改的胞嘧啶使用基础切除修复途径11。
人类TET2基因被确定为在不同的造血恶性肿瘤, 包括骨髓增生异常综合征 (mds)14,15,16, mds-骨髓增生性肿瘤经常突变的基因 (mds-mpn) 和急性髓系白血病 (AML) 源自 mds 和 mds-MPN16。与野生型 (wt) TET214相比, TET2 突变患者骨髓 DNA 的5hmC 修饰水平较低。一些小组已经开发出 TET2-knockout 小鼠模型, 以阐明其在正常造血和髓样转化中的作用17,18,19,20。这些在 TET2 基因突变的小鼠最初是正常和可行的, 但表现出不同的造血恶性肿瘤, 因为他们的年龄导致他们的早期死亡。这些研究表明 wt-TET2 在正常造血分化中发挥的重要作用。在这些小鼠模型中, 杂合造血干细胞 (TET2+/造血干细胞) 和纯合 TET2造血干细胞在 wt-TET2 造血谱系中具有优于纯合造血干细胞重新填充的竞争优势, 同时 TET2TET2 造血干细胞开发了多种造血恶性肿瘤17,18。这些研究表明, TET2 酶的 haploinsufficiency 改变了造血干细胞的发展, 导致了造血恶性肿瘤。
与 TET2 基因突变的小鼠相似, 大多数白血病患者 haploinsufficiency TET2 酶活动的表现。这些主要杂合体细胞突变包括帧移位和废话突变分散在整个 TET2 基因体内, 而错义突变是最聚集在酶领域12。到目前为止, 文献中很少有关于 wt 和 mutant-TET2 的表征, 主要是由于 TET2 酶的生产和测定21的困难。在这里, 我们报告了一个简单的单步纯化原生 TET2 酶使用离子交换色谱法。进一步, 对 TET2 酶的酶活性进行了优化, 并进行了定量的 LC-ms/ms 测定。

<table border="0" cellpadding="0" cellspacing="0" style="width:739px;" width="739">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">HEPES</td>
			<td style="width:201px;">Carbosynth</td>
			<td style="width:119px;">FH31182</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Iron(II) sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>F8633</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">&#945;-Ketoglutaric acid (2-Oxoglutaric acid)</td>
			<td>Sigma-Aldrich</td>
			<td>K1750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">L-Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
			<td style="width:201px;">Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Ammonium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>A1542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">HPLC grade water</td>
			<td>Fisher Scientific</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Oligo clean and concentrator</td>
			<td>Zymo Research</td>
			<td>D4061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">DNAse I</td>
			<td>New England Biolabs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">S1 Nuclease</td>
			<td>Thermo Scientific</td>
			<td>ENO321</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">CIAP (Calf intestinal alkaline phosphatase)</td>
			<td>New England Biolabs</td>
			<td>M0290S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">LB Media</td>
			<td>Affymetrix</td>
			<td>J75852</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">IPTG</td>
			<td>Carbosynth</td>
			<td>EI05931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">MES [2-(N-Morpholino)ethanesulfonic acid monohydrate]</td>
			<td style="width:201px;">Carbosynth</td>
			<td>FM37015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7893</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">SP Sepharose</td>
			<td>Fisher Scientific</td>
			<td>45-002-934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxy-5-methylcytidine</td>
			<td>TCI</td>
			<td>D3610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxy-5-hydroxymethyalcytidine</td>
			<td>TCI</td>
			<td>D4220</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">2&#39;-Deoxycytidine-5-carboxylic acid, sodium salt</td>
			<td>Berry &#38; Associates</td>
			<td>PY 7593</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">5-Formyl-2&#39;-deoxycytidine</td>
			<td>Berry &#38; Associates</td>
			<td>PY 7589</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxycytidine</td>
			<td>Berry &#38; Associates</td>
			<td>PY 7216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxyadenosine</td>
			<td>Carbosynth</td>
			<td>ND04011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxyguanosine</td>
			<td>Carbosynth</td>
			<td>ND06306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxythymidine</td>
			<td>VWR Life Science</td>
			<td>97061-764</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Gateway technology</td>
			<td style="width:201px;">Thermo Fisher</td>
			<td style="width:119px;">11801016</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:252px;">Beckman Allegra X-15R centrifuge&#160;</td>
			<td style="width:201px;">Beckman Coulter</td>
			<td style="width:119px;">392932</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Sonic Dismembrator 550</td>
			<td style="width:201px;">Fisher Scientific</td>
			<td style="width:119px;">XL2020</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">&#196;KTA FPLC system</td>
			<td style="width:201px;">Pharmacia (GE Healthcare)</td>
			<td style="width:119px;">18116468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">FreeZone 4.5 freeze dry system</td>
			<td style="width:201px;">Labconco</td>
			<td style="width:119px;">7750020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Zymo Oligo purification columns&#160;</td>
			<td style="width:201px;">Zymo Research</td>
			<td style="width:119px;">D4061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">BDS Hypersil C18 column</td>
			<td style="width:201px;">Keystone Scientific, INC</td>
			<td style="width:119px;">105-46-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">3200 Q-Trap mass spectrometer</td>
			<td style="width:201px;">AB Sciex</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">HPLC&#160;</td>
			<td style="width:201px;">Shimadzu HPLC&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">XK16/20 FPLC column</td>
			<td style="width:201px;">Pharmacia (GE Healthcare)</td>
			<td style="width:119px;">28988937</td>
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		</tr>
	</tbody>
</table>

efficient purification and lc-ms/ms-based assay development for ten-eleven translocation-2 5-methylcytosine dioxygenase

5-甲基胞嘧啶 (5mC) 介导的表观遗传转录调控在真核发育中起着至关重要的作用。这些表观遗传标记的去甲基化是通过顺序氧化十-十一易位芳香烃 (TET1-3), 其次是胸腺嘧啶 DNA glycosylase 依赖基础切除修复。由于基因突变或其他表观遗传机制, TET2 基因的失活与不同癌症, 特别是造血恶性肿瘤患者预后较差有关。在这里, 我们描述了一个有效的单步纯化酶活性无标签人 TET2 酶使用阳离子交换层析。我们进一步提供液相色谱-串联质谱 (LC ms/ms) 方法, 可以分离和量化四个正常的 DNA 基础 (a, T, G 和 C), 以及四个改性胞嘧啶碱基 (5-甲基, 5-羟甲基, 5-甲酰基, 和 5-羧基)。本试验可用于评价野生型和突变 TET2 芳香烃的活性。

芳香烃5mC 在 DNA 中的动态修饰在表观遗传转录规则中起着重要作用。TET2 酶在不同的造血恶性肿瘤12经常变异。为了研究 TET2 酶在正常发育和疾病中的作用, 我们克隆了其最小催化活性域, 而没有任何亲和标记进入 pDEST14 向量22。在细菌大肠杆菌BL21 (DE3) 细胞中, 通过 SDS-页分析, 在∼5% 的总可溶性蛋白中生成无标记 TET2 酶。由于 TET2 ?...

Watch this Scientific Journal Video about Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase at JoVE.com

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

在这里, 我们提出了一个有效的单步纯化活性无标签人的协议十-十一 translocation-2 (TET2) 5-甲基胞嘧啶酶使用离子交换色谱法及其测定液相色谱-串联质量基于质谱 (LC-ms) 的方法。

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

该方法有助于回答表观遗传学和TET2中西5-甲基细胞素脱甲基化领域中的关键问题，例如对正常和临床TET2突变活性的分析。该技术的主要优点是，原生TET2可以在一个步骤中进行纯化，并且可以从所有三种产品的形成的角度分析其活性。几位研究生将演示这个程序。蛋白质纯化将由查扬·巴塔查里亚、阿宁达·桑达里·戴伊的TET2测定和纳维德·阿永的LC-MS/MS分析进行。对于细菌转化，在1.7毫升管中加入含有未标记人类TET2二二氧酶的重组pDEST 14目标表达载体的微升至100微升的化学能力大肠杆菌BL21 DE3细胞中。在冰上孵育至少15分钟后，在42摄氏度的温度下将混合物在水浴中加热30秒。热休克后立即将细胞放回冰上至少两分钟。在此之后，加入250微升的超优肉汤与代谢压抑细胞。在37摄氏度的摇床中孵育细菌细胞一小时。孵育后，在9000倍重力下将管子离心一分钟，将细胞旋转下来。通过移液丢弃70%的上流液，并在剩余的介质中溶解颗粒。将细胞悬浮液铺在每毫升安西林含有100微克的利鲁阿肉汤加糖板上。在37摄氏度下孵育板16小时。选择一个孤立的菌落，并接种到10毫升的LB安西林介质。在摇床中孵育管在37摄氏度过夜。在此之后，使用100微升细菌培养剂接种100毫升LB安西林培养基作为主要培养基。在摇床中孵育管在37摄氏度过夜。第二天，接种15个烧瓶，每个含有600毫升的LB安西林介质，每个烧瓶有6毫升的原培养。在转速为 180 RPM 的摇床上，在 37 摄氏度的温度下孵化烧瓶。要检查细菌培养的密度，请使用分光光度计测量其光学密度为 600 纳米（OD600）。在OD600时培养达到0.8的密度后，在每个烧瓶中用300微升的一摩尔IPTG诱导TET2蛋白的表达，并在17摄氏度下再培养16小时。孵育后，将细菌培养转移到离心瓶中。将表达 TET2 酶的细菌培养力在 5，250 倍重力下离心 45 分钟。使用细菌颗粒进行 TET2 纯化。在冰上或在4摄氏度下工作，将细菌颗粒重新悬浮在100毫升50毫升的 MES 缓冲液缓冲液中，pH6。然后服用5毫升的细胞悬浮液，并把它带到45毫升与缓冲液。在电源 20 下，以 60 秒的冷却间隔将悬架进行 5 次 30 秒的声波化。在重力下旋转裂酸45分钟。收集含有可溶性 TET2 酶的上清液，在加载到 FPLC 系统之前，通过 0.45 微米过滤器。将 30 毫升的强阳离子交换树脂包装成 FPLC 柱。使用 FPLC 系统以每分钟 0.3 毫升的恒定流量将柱与 10 床洗涤缓冲液平衡。将澄清的 lysate 加载到预平衡柱上，用大约 10 床的洗涤缓冲液清洗，直到流经变得清晰。Elute TET2 使用 15 个床体积中从洗涤缓冲液到洗涤缓冲液的 0 到 10% 梯度，然后是两个床体积的 10% 洗脱缓冲液的保持。收集100微升细胞糖酸盐样品之前和之后列加载，连同所有的洗脱分数，并分析10%解析SDS-PAGE凝胶。将含有 TET2 蛋白质的分数集中，冷冻干燥蛋白质。然后将酶溶解在10毫升水中，储存在零下80摄氏度。使用三微克基板在三钙中执行所有脱甲基反应。在总反应缓冲液的50微升中加入100微克纯化的TET2酶。在37度下孵育一小时后，用5个微升500毫升EDTA的催化氧化反应淬火。为了准备样品进行分析，首先在55微升淬火反应中加入100微升的Oligo结合缓冲液，将DNA从TET2反应混合物中分离。在此之后，在混合物中加入400微升100%乙醇。通过 Oligo 绑定列传递此混合物。用750微升洗涤缓冲液清洗绑定DNA后，在20微升水中洗脱DNA。现在，在37摄氏度下用两个DNase I和60个单位的S1核酸酶消化分离的DNA，12小时，产生单个核苷单磷酸盐。消化后，在样品中加入两个单位的小牛肠道碱性磷酸酶。在37摄氏度下孵育12小时，从核苷单磷酸盐中去除末期磷酸盐组，并获得核苷酸。为LC-MS/MS方法的开发准备所有改性细胞素核苷的100微摩尔存量溶液，以及HPLC级水中的正常DNA碱基。在增强的 MS 扫描模式下，在质谱仪中逐次注入库存解决方案，以每分钟 10 微升的流速，优化核苷依赖 MS/MS 参数。几个参数可以使用软件的自动定量优化功能针对每个DNA核苷进行优化。此时，使用流注入分析模式下软件的手动定量优化功能优化每个DNA核苷的剩余参数。现在，使用 25%溶剂 B 的梯度注入 10 微升库存溶液，以每分钟 0.3 毫升的流速，优化依赖源的 MS/MS 参数。要分离所有八个 DNA 核苷酸，请执行 C18 列文本协议中详细说明的液相色谱。最后，在标准曲线中使用LC-MS/MS方法检测和量化所有核苷酸。由于与大多数本地大肠杆菌蛋白相比，TET2的催化领域具有相对较高的等电点，因此开发出了利用阳离子交换色谱进行高效纯化工艺。这种纯化在一步中产生超过90%的纯TET2酶。为了分离和量化不同的脱氧核糖核酸衍生物和TET2酶反应后四个天然DNA碱基，对基于LC-MS/MS的敏感测定进行了优化。标准曲线是使用含有所有核苷的混合物的序列稀释法绘制的。此处显示了液相色谱 MS/MS 方法的结果。该方法允许分离和定量四个正常的DNA碱基，以及四个修改的细胞氨酸碱。在这个实验过程中，我们描述了使用位点特异性重组技术和在E.Coli的目的地载体中充分表达未标记的人类TET2二氧酶催化域的克隆。我们利用阳离子交换色谱有效纯化未标记的 TET2 酶，在一步中产生超过 90% 的纯度。此外，我们开发了一种新的液相色谱法，分离出四个正常的DNA碱基和四个改性细胞氨酸碱。为了量化 TET2 催化反应中的八个核苷酸，我们结合我们改进的液相色谱方法与串联质谱法。然后利用敏感的LC-MS/MS测定来确定重组未标记的人类TET2酶的活性。此处描述的方法将大大提高野生型和突变的 TET2 二氧酶的估值。

Research

JoVE Journal

Biochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential Scanning Calorimetry ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

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 ...

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 ...

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

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, ...

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 ...

Ganglioside Extraction, Purification and Profiling

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...