这项工作得到了 Politécnico 国立 Secretaria de Investigación y Posgrado (IPN-SIP-20170864)、方案 de 支持农场 Proyectos-Investigación e Innovación Tecnológica (PAPIIT IN222117)-国立大学国立墨西哥的赠款的支持 (UNAM) 和委员会 Ciencia y Tecnología (CONACyT 254904-JPP 和 256520-GGS)。Fundação de 宪法保护 Pesquisa 做里约热内卢 (FAPERJ Cientistas 做 Nosso: E 26/103.353/2011)。我们感谢 QFB。奥斯卡豪尔赫·伊万·莫拉·戈多伊 Luqueño Bocardo 为示意图概述。我们感谢米格尔博士 Tapia 罗德里格斯在 LDs 的共焦显微术中的宝贵帮助。我们要感谢布鲁诺 Bozaquel Morais 博士在开发酵母技术方面的开创性工作, 我们已经适应了U. 玉米。

1. 缓冲和解决方案的准备工作
<ol><li>制备 1 L 磷酸缓冲盐水 (PBS, pH 值 7), 溶解8克氯化钠, 0.2 克氯化钾, 1.44 克 Na2HPO4, 0.24 g 的馏分2PO4在800毫升的水。用 HCl 将 pH 值调整为 7.0, 然后在1升的最终容积中加水. PBS 可作为10x 库存解决方案, 并存储在室温下。</li>
	<li>要准备10毫升的固定溶液, 添加1毫升37% 甲醛到9毫升的 PBS, 达到最后浓度的3.7%。请在使用前准备此解决方案。 注意: 使用手套处理甲醛溶液。</li>
	<li>为制备淬火溶液 (500 毫米), 在100毫升蒸馏水中溶解8.3 克。在使用前准备此解决方案, 并将其存储在室温下。</li>
	<li>准备 BODIPY 10 毫米的溶液, 溶解10毫克的 BODIPY 493/503 在3.8 毫升的二甲基亚砜 (亚砜)。保持100µL 整除数在黑暗中, 在-70 摄氏度。</li>
	<li>要准备 BODIPY 5 µM 淬火溶液, 添加1µL 10 毫米 BODIPY 493/503 至2毫升的500毫米淬火溶液。对于 LFR 化验, 需要最终浓度为5µM 的 BODIPY。</li>
	<li>要准备矿物溶液, 请添加0.06 克 H3BO3, 0.14 克 MnCl2-4H2O, 0.4 g ZnCl2, 0.04 g 的 Na2MoO42H2o, 0.1 g FeCl36H2o 和 0.4 g 丘索4-5H2O 到1升蒸馏水。</li>
	<li>准备盐溶液, 添加 16 g 的2PO4, 4 克的 Na2所以4, 8 g 的氯化钾, 2 克 MgSO4, 1 克的 CaCl2, 和8毫升的矿物溶液的 1 L 的蒸馏水。</li>
	<li>为准备无氮源的最小培养基用于U. 玉米 FB2 (a2b2),将10克葡萄糖和62.5 毫升盐溶液添加到蒸馏水的1升, 根据节假日, et等。31. 将 pH 值调整为 7.0, 然后在250毫升烧瓶中分配100毫升的培养基, 并对其进行消毒。在液体循环或过滤灭菌时, 在 15 psi (1.05 公斤/厘米2) 上的高压釜为20分钟。</li>
	<li>制备酵母蛋白胨葡萄糖培养基 (YPD), 添加5克的酵母萃取物, 2.5 克的蛋白胨, 5 克葡萄糖到1升的蒸馏水。在250毫升烧瓶中分配100毫升溶液, 并对其进行消毒。在液体循环或过滤灭菌时, 在 15 psi (1.05 公斤/厘米2) 上的高压釜为20分钟。</li>
</ol>2. 培养条件和细胞固定
注意: 通过尽快修复单元格来保留 LDs 的结构。可在离心管内进行固定。固定的细胞可以保持在摄氏4摄氏度, 一个月, 如果脱水是避免留下一层薄薄的水。
<ol><li>在与该研究相关的文化条件下生长玉米细胞。启动具有初始 OD600 (1.15 x 106单元格/mL) 的区域性。孵化细胞在28°c, 180 rpm 24 小时。OD600的1对应于 2.3 x 107单元格/mL。</li>
	<li>在选定的时间, 撤回单元格的整除。对于U. 玉米,在前8小时内每2小时提取5毫升。经过8小时的生长, 整除数2毫升就足够了。</li>
	<li>测量每整除 600 nm 的光学密度。</li>
	<li>用台式离心机将细胞悬浮在 1.4万 x g 处以1分钟为4摄氏度。放弃上清。</li>
	<li>在1毫升的 PBS 甲醛3.7% 缓冲器中悬浮细胞的颗粒。室温下孵化细胞15分钟。</li>
	<li>孵化后, 离心机的细胞悬浮在 1.4万 x g 1 分钟4摄氏度。放弃上清。</li>
	<li>用类似的蒸馏水量 (1 毫升) 清洗细胞颗粒两次。离心机细胞悬浮在 1.4万 x g 1 分钟在4°c。每次洗涤步骤后都要丢弃上清液。</li>
	<li>用蒸馏水 (等式 8) 将单元颗粒调整为 5 OD600 (1.15 x 101细胞/毫升)。 <img alt="Equation 1" src="/files/ftp_upload/57279/57279eq1.jpg">    等式1。
</li>
	<li>保持样品在4摄氏度, 直到它的使用。</li>
</ol>3. 液体荧光恢复试验 (LFR)
<ol><li>打开分光光度计 Skanlt 软件。单击新会话, 然后选择开始, 然后Varioskan 莱克丝。从会话树中选择协议, 输入荧光波长的设置 (激发 485 nm/发射 510 nm; 光学密度 600 nm) 并选择自动光电倍增管增益。对于励磁带宽, 请选择 12 nm。在光学中, 选择顶部 (样品的激发是从井的顶部)。</li>
	<li>进入一个温和和连续的鼓动 (300 转每分钟)。选择 "运行板出",暂停直到用户操作(用于将示例添加到油井的时间)。重复该协议五次。单击保存, 并在会话名称字段上为会话编写名称。该协议现在已准备好进行样本分析。</li>
	<li>将 BODIPY 5 µM 淬火液的200µL 添加到96井黑底板的每一个井中。</li>
	<li>将盘子放在分光光度计室内, 在30摄氏度孵育5分钟。从这一点上, 尽可能多地保护盘子不受光线的侵害。</li>
	<li>单击开始按钮, 并读取与空白对应的荧光 (激发 485/发射 510) 和 OD600 。</li>
	<li>在暂停时间内, 将甲醛固定细胞悬浮液的5µL 添加到井中。用吸管仔细地混合样品。将盘子放在分光光度计内, 然后单击继续。然后, 将根据上面设计的协议处理样品。</li>
	<li>重复三连续增加的5µL 细胞悬浮。确保细胞不会沉淀。(图 1)。</li>
</ol>4. LD 指数的计算
<ol><li>对于微板块中的每个样本, 绘制一个荧光和吸光度的图, 对每一个连续加法 (包括空白的5点) 的体积。在本研究中, 使用 Microsoft Excel 软件进行计算。要绘制数据, 请分别在数据表的第一、第二和第三列中输入卷、荧光和吸光度数据。然后, 使用游标选择整个数据, 单击 "插入并选择 (XY) 散点"。</li>
	<li>为图表中的每一行选择点, 并在选定的显示公式框中插入相应的趋势线。根据等式 2, 写下两条直线的斜率值: <img alt="Equation 2" src="/files/ftp_upload/57279/57279eq2.jpg">    等式2 其中 y = 荧光或光密度, x = 样本量 (0、5、10、15、20µL)、 m = 荧光或光密度线的斜率, 以及 b = y 截距。</li>
	<li>将荧光线的斜率除以光密度线的斜率, 得到 LD 指数, 方程3。 <img alt="Equation 3" src="/files/ftp_upload/57279/57279eq3.jpg">    等式3 LD 指数对应于荧光和光密度斜率的商。</li>
</ol>5. 数据质量分析
<ol><li>计算每条直线的相关系数: 单击函数向导(fx), 然后选择CORREL。如果 r < 0.9, 丢弃数据并重复读数。如果 r ≥ 0.9, 读数是可靠的。</li>
</ol>

<ol>
	<li>Guo, Y., Cordes, K. R., Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+at+a+glance.">Lipid droplets at a glance.</a> Journal of Cell Science. 122 (6), 749-752 (2009).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+roles+for+lipid+droplets.">Expanding roles for lipid droplets.</a> Current Biology. 25 (11), R470-R481 (2015).</li><li>Sestric, R., Munch, G., Cicek, N., Sparling, R., Levin, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+neutral+lipid+synthesis+by+Yarrowia+lipolytica+on+various+carbon+substrates+under+nutrient-sufficient+and+nutrient-limited+conditions.">Growth and neutral lipid synthesis by Yarrowia lipolytica on various carbon substrates under nutrient-sufficient and nutrient-limited conditions.</a> Bioresource Technology. 164, 41-46 (2014).</li><li>Raimondi, 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=Getting+lipids+from+glycerol:+new+perspectives+on+biotechnological+exploitation+of+Candida+freyschussii.">Getting lipids from glycerol: new perspectives on biotechnological exploitation of Candida freyschussii.</a> Microbial Cell Factories. 13, 83 (2014).</li><li>Cescut, J., Fillaudeau, L., Molina-Jouve, C., Uribelarrea, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+accumulation+in+Rhodotorula+glutinis+induced+by+nitrogen+limitation.">Carbon accumulation in Rhodotorula glutinis induced by nitrogen limitation.</a> Biotechnology for Biofuels. 7, 164 (2014).</li><li>Galafassi, 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=Lipid+production+for+second+generation+biodiesel+by+the+oleaginous+yeast+Rhodotorula+graminis.">Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis.</a> Bioresource Technology. 111, 398-403 (2012).</li><li>Kot, A. M., Blazejak, S., Kurcz, A., Gientka, I., Kieliszek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodotorula+glutinis-potential+source+of+lipids,+carotenoids,+and+enzymes+for+use+in+industries.">Rhodotorula glutinis-potential source of lipids, carotenoids, and enzymes for use in industries.</a> Applied Microbiology and Biotechnology. 100 (14), 6103-6117 (2016).</li><li>Rakicka, M., Lazar, Z., Dulermo, T., Fickers, P., Nicaud, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+production+by+the+oleaginous+yeast+Yarrowia+lipolytica+using+industrial+by-products+under+different+culture+conditions.">Lipid production by the oleaginous yeast Yarrowia lipolytica using industrial by-products under different culture conditions.</a> Biotechnology for Biofuels. 8, (2015).</li><li>Zhu, Z. W., 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=Dynamics+of+the+Lipid+Droplet+Proteome+of+the+Oleaginous+Yeast+Rhodosporidium+toruloides.">Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides.</a> Eukaryotic Cell. 14 (3), 252-264 (2015).</li><li>Kolouchova, I., Mat'atkova, O., Sigler, K., Masak, J., Rezanka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Palmitoleic+and+Linoleic+Acid+in+Oleaginous+and+Nonoleaginous+Yeast+Biomass.">Production of Palmitoleic and Linoleic Acid in Oleaginous and Nonoleaginous Yeast Biomass.</a> International Journal of Analytical Chemistry. , (2016).</li><li>Radulovic, 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=The+emergence+of+lipid+droplets+in+yeast:+current+status+and+experimental+approaches.">The emergence of lipid droplets in yeast: current status and experimental approaches.</a> Current Genetics. 59 (4), 231-242 (2013).</li><li>Takahashi, Y., 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=Perilipin-Mediated+Lipid+Droplet+Formation+in+Adipocytes+Promotes+Sterol+Regulatory+Element-Binding+Protein-1+Processing+and+Triacylglyceride+Accumulation.">Perilipin-Mediated Lipid Droplet Formation in Adipocytes Promotes Sterol Regulatory Element-Binding Protein-1 Processing and Triacylglyceride Accumulation.</a> Plos One. 8 (5), e64605 (2013).</li><li>Thiam, A. R., Farese Jr, R. V., Walther, T. 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+biophysics+and+cell+biology+of+lipid+droplets.">The biophysics and cell biology of lipid droplets.</a> Nature Reviews Molecular Cell Biology. 14 (12), 775-786 (2013).</li><li>Penno, A., Hackenbroich, G., Thiele, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipids+and+lipid+droplets.">Phospholipids and lipid droplets.</a> Biochimica et Biophysica Acta. 1831 (3), 589-594 (2013).</li><li>Ageitos, J. M., Vallejo, J. A., Veiga-Crespo, P., Villa, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oily+yeasts+as+oleaginous+cell+factories.">Oily yeasts as oleaginous cell factories.</a> Applied Microbiology and Biotechnology. 90 (4), 1219-1227 (2011).</li><li>Athenstaedt, K., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+neutral+lipids:+synthesis,+storage+and+degradation.">The life cycle of neutral lipids: synthesis, storage and degradation.</a> Cellular and Molecular Life Sciences. 63 (12), 1355-1369 (2006).</li><li>Marshall, L. L., Stimpson, S. E., Hyland, R., Coorssen, J. R., Myers, 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=Increased+lipid+droplet+accumulation+associated+with+a+peripheral+sensory+neuropathy.">Increased lipid droplet accumulation associated with a peripheral sensory neuropathy.</a> Journal of Biological Chemistry. 7 (2), 67-76 (2014).</li><li>Filipe, A., McLauchlan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+and+lipid+droplets:+finding+a+niche.">Hepatitis C virus and lipid droplets: finding a niche.</a> Trends in Molecular Medicine. 21 (1), 34-42 (2015).</li><li>Greenberg, A. 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=The+role+of+lipid+droplets+in+metabolic+disease+in+rodents+and+humans.">The role of lipid droplets in metabolic disease in rodents and humans.</a> Journal of Clinical Investigation. 121 (6), 2102-2110 (2011).</li><li>Aguilar, L. R., 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=Lipid+droplets+accumulation+and+other+biochemical+changes+induced+in+the+fungal+pathogen+Ustilago+maydis+under+nitrogen-starvation.">Lipid droplets accumulation and other biochemical changes induced in the fungal pathogen Ustilago maydis under nitrogen-starvation.</a> Archives of Microbiology. , (2017).</li><li>Bozaquel-Morais, B. L., Madeira, J. B., Maya-Monteiro, C. M., Masuda, C. A., Montero-Lomeli, 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+new+fluorescence-based+method+identifies+protein+phosphatases+regulating+lipid+droplet+metabolism.">A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.</a> PLoS One. 5 (10), e13692 (2010).</li><li>Madeira, J. B., 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=TORC1+Inhibition+Induces+Lipid+Droplet+Replenishment+in+Yeast.">TORC1 Inhibition Induces Lipid Droplet Replenishment in Yeast.</a> Molecular and Cellular Biology. 35 (4), 737-746 (2015).</li><li>Maya-Monteiro, C. 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=Leptin+induces+macrophage+lipid+body+formation+by+a+phosphatidylinositol+3-kinase-+and+mammalian+target+of+rapamycin-dependent+mechanism.">Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism.</a> Journal of Biological Chemistry. 283 (4), 2203-2210 (2008).</li><li>Listenberger, L. L., Brown, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Cell Biology. , (2001).</li><li>Jump, D. B., Torres-Gonzalez, M., Olson, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soraphen+A,+an+inhibitor+of+acetyl+CoA+carboxylase+activity,+interferes+with+fatty+acid+elongation.">Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation.</a> Biochemical Pharmacology. 81 (5), 649-660 (2011).</li><li>Hobro, A. J., Smith, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li><li>Chen, Y. T., Wan, L., Zhang, D. P., Bian, Y. Z., Jiang, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+spectroscopic+property+of+Bodipy+derivates+through+tuning+the+molecular+configuration.">Modulation of the spectroscopic property of Bodipy derivates through tuning the molecular configuration.</a> Photochemical &amp; Photobiological Sciences. 10 (6), 1030-1038 (2011).</li><li>Pagac, 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=SEIPIN+Regulates+Lipid+Droplet+Expansion+and+Adipocyte+Development+by+Modulating+the+Activity+of+Glycerol-3-phosphate+Acyltransferase.">SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase.</a> Cell Reports. 17 (6), 1546-1559 (2016).</li><li>Qiu, B., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BODIPY+493/503+Staining+of+Neutral+Lipid+Droplets+for+Microscopy+and+Quantification+by+Flow+Cytometry.">BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry.</a> Bio-protocol. 6 (17), (2016).</li><li>Rumin, 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=The+use+of+fluorescent+Nile+red+and+BODIPY+for+lipid+measurement+in+microalgae.">The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae.</a> Biotechnology for Biofuels. 8, 42 (2015).</li><li>Holliday, R., King, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ustilago+maydis.">Ustilago maydis.</a> Handbook of genetics. , 575-595 (1974).</li></ol>

作者没有什么可透露的

LFR 是首次应用于研究酵母中脂质代谢的新方法, 后来在U. 玉米20,21中成功实现。虽然 ld 指数并没有给出细胞中累积的标记的绝对值, 但它为细胞的脂质含量提供了一个快速的概念, 当比较两个或多个实验条件的 LD 指数时, 对数据的解释是直截了当的。BODIPY 493/503 是高度特异的中性脂, 主要成分的 LDs, 它有一个狭窄的发射频谱, 以促进同时检测...

脂滴 (LDs) 是无处不在的细胞内脂肪体组成的核心中性脂, 主要是标签和甾醇酯 (SE)。核心周围有一层磷脂, 与蛋白质如 perilipin 和酶参与合成中性脂, 如甘油酰基转移酶, 乙酰基 coa 酶和酰 coa 综合体,1。由于其动态行为, 磷脂单层还含有水解标记和 SE2的三酰甘油脂肪酶。根据细胞类型和有机体, 储存中性脂可以用来产生能量, 或合成磷脂和信号分子。在酵母和其他真菌中, LDs 的含量随培养基中氮/碳比值的变化而变化, 表明氮的可用性降低可能是提高中性脂产量的关键3,4 ,5。在酵母中生产大量的标签, 在生物技术中作为生物燃料的来源和食品工业中有潜在的用途。一些储存的脂质含有高比例的多不饱和脂肪酸, 如欧米茄3和欧米茄 6, 营养和膳食重要性6,7,8,9,10,11. 包括人类细胞在内的哺乳动物细胞的 LDs 也含有标记和胆固醇酯。磷脂单层与酵母 LDs 中描述的蛋白质的哺乳动物同源性相互作用, 另外一种蛋白质, perilipin, 在酵母12中不存在。磷脂单层及其相关蛋白的一个建议作用是稳定 lds 结构, 并允许 lds 与细胞器的相互作用, 如线粒体、内质网、过氧化物酶体和液泡, 主要用于脂交换13,14。有趣的是, 在人类中, LDs 似乎参与了2型糖尿病, 动脉粥样硬化, 脂肪性肝炎和冠心病的病理, 其中有增加的数字15,16,17.某些类型的病毒使用 LDs 作为平台来组装病毒2,18,19。
由于 lds 在人类病理中的影响及其潜在的生物技术应用, 对 lds 形成的精确实验测定是一项重要的任务。本文描述了一个可靠的检测方法的基础上恢复荧光 (LFR) 的 BODIPY 493/503 (4, 4-difluoro-1, 3, 5, 7, 8-pentamethyl-4-波拉-3 a, 4 a-diaza s indacene), 以获得相对价值的中性脂在细胞中的含量。通过这种分析, 可以跟踪真菌中中性脂类积累的动态, 如U. 玉米和酿酒酵母, 还有哺乳动物细胞, 不需要脂质提取20.该方法首次在酿酒酵母中应用, 以确定参与调节脂质代谢的蛋白磷酸和激酶。这是可能的, 不用油脂提取或蛋白质纯化21,22。LFR 也被用来建立腹腔巨噬细胞的 LDs 形成的动力学23。BODIPY 493/503 的使用比其他中性脂质染料有一定的优势, 如尼罗河红色。BODIPY 493/503 是高度特异的中性脂质和有一个狭窄的放射频谱, 促进同时检测的信号, 如红色荧光蛋白或图面-跟踪, 当样品分析的共焦显微镜。不幸的是, BODIPY 493/503 对漂白很敏感, 但在暴露于光24中使用 antiquenching 试剂可以避免此过程。
在酵母细胞中进行 LFR 测定, 在理想的营养条件下培养, 整除数在不同时间被提取。其次, 细胞是固定的甲醛, 这保持了 LDs 的完整性数月, 当细胞存储在4摄氏度。其他固定技术应避免, 特别是使用甲醇或冷丙酮, 因为它们导致 LDs 在细胞内的退化24。为了测量 LD 指数, 甲醛固定细胞悬浮在水中, 以获得明确的浓度。然后, 它们被添加到包含荧光 BODIPY 493/503 的解决方案中, 当荧光进入细胞并与 LDs 关联时, 它的荧光就会恢复。伴随荧光测量 (485 nm/510 nm), 细胞的浓度通过测量 600 nm 的光学密度来量化。每一个样品被读四次通过增加随后5µL 整除数甲醛固定的细胞悬浮到同样井。荧光和吸光度的空白在细胞的加法之前获得。通过确定测量的线性度来评估荧光和吸光度数据的质量: 如果 r < 0.9, 数据被丢弃。r 值是重要的, 因为基本上荧光强度应该产生一个线性反应, 以增加细胞浓度。如果细胞浓度过高, 线性度就会丢失。LFR 法为高通量实验中选择所需的 LD 表型提供了一种快速、简便、经济的方法。在选择所需条件后, 可以通过共焦显微镜对单个细胞的 LD 含量进行研究, 使用与 BODIPY 染色的相同的甲醛固定细胞, 提供细胞中 LDs 的图像。用薄层色谱法进一步分析了它们的标记和硒含量。

<table>
	<tbody>
		<tr>
			<td>Spectrophotometer</td>
			<td>Varioskan Lux multimode microplate reade</td>
			<td>D5879</td>
			<td>Filter 493/503 or monochromator detector</td>
		</tr>
		<tr>
			<td>Plate costar</td>
			<td>Corning Inc.</td>
			<td>3615</td>
			<td>96 well black-wall/clear bottom</td>
		</tr>
		<tr>
			<td>Plate costar&#160;</td>
			<td>Corning Inc</td>
			<td>3598</td>
			<td>96 well cell culture plate costar</td>
		</tr>
		<tr>
			<td>BODIPY 493/503</td>
			<td>Invitrogen/ Thermofisher</td>
			<td>D3922</td>
			<td>4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene</td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma Aldrich</td>
			<td>252549</td>
			<td>ACS reagent, 37 wt % in H2O, contains 10-15% methanol to prevent polymerization</td>
		</tr>
		<tr>
			<td>Potassium iodide</td>
			<td>Sigma Aldrich</td>
			<td>60399</td>
			<td>BioUltra, &#8805; 99.5% (AT)</td>
		</tr>
		<tr>
			<td>FB2 Ustilago maydis</td>
			<td>ATCC</td>
			<td>201384</td>
			<td>Basidiomycete-Yeast</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>746398</td>
			<td>ACS reagent, inorganic salt</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P0662</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>Select Yeast Extract</td>
			<td>Sigma Aldrich</td>
			<td>Y100</td>
			<td>Mixture of amini acids, peptides, water- soluble, vitamins and carbohydrates for culture media</td>
		</tr>
		<tr>
			<td>N-Z case plus</td>
			<td>Sigma aldrich</td>
			<td>N4642</td>
			<td>Casein enzymatic hydrolyzate from bovine milk</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G-8270</td>
			<td>D (+) glucose</td>
		</tr>
		<tr>
			<td>Skaker flask</td>
			<td>Pirex</td>
			<td>CLS4450250-6EA</td>
			<td>Borosiicate glass</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>SEV M&#233;xico</td>
			<td>INO650V-7</td>
			<td>Orbital shaker</td>
		</tr>
		<tr>
			<td>Centrifuge table</td>
			<td>Eppendorf</td>
			<td>5415C</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Sigma Aldrich</td>
			<td>Z606340</td>
			<td>Eppendorf</td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Axygen scientific</td>
			<td>T-200-Y</td>
			<td>Universal Pipet Tips with Bevelled End, 200 microliter, non sterile</td>
		</tr>
		<tr>
			<td>mLine pipette</td>
			<td>Biohit</td>
			<td>725130</td>
			<td>8 channels, volume range 5-100 uL</td>
		</tr>
	</tbody>
</table>

lipid index determination by liquid fluorescence recovery in the fungal pathogen ustilago maydis

本文介绍了应用 LD 指数法测定脂滴 (LDs) 中甘油三酯 (微板块) 积累的灵敏的检测方法。获得了不带脂质提取的 LD 指数。它允许测量在不同条件下的高吞吐量实验中的 LDs 含量, 如富含或氮气耗尽介质的生长。尽管该方法首次被描述为研究酿酒酵母中的脂滴代谢, 它成功地应用于担子黑穗病玉米。有趣的是, 由于 LDs 是真核细胞 phylogenetically 保存的细胞器, 这种方法可以应用于大量的不同的干细胞, 从酵母到哺乳动物细胞。LD 指数是基于 BODIPY 493/503 在淬火条件下的液体荧光恢复试验 (LFR), 通过添加与甲醛固定的细胞.碘钾用作荧光淬火。荧光与光密度斜率的比值为 LD 指数。斜坡是计算的直线, 当 BODIPY 荧光和光学密度在 600 nm (OD600) 绘制的样本添加。最佳数据质量是由相关系数等于或高于 0.9 (r ≥ 0.9) 反映出来的。可以同时读取多个示例, 因为它可以在微板块中实现。由于 BODIPY 493/503 是一种脂溶性荧光染料, 分成脂肪滴, 它可以用于许多类型的细胞积累 LDs。

LFR, 与 BODIPY 493/503 作为荧光探针是一个可靠和容易的方法来研究 LDs 在U. 玉米,的动态积累, 无论生长条件如何。当细胞在 YPD 培养基中培养时, LD 指数在指数阶段增加, 其次是固定相的下降 (图 2A)。与此相反, 在氮饥饿下生长的细胞中, LD 指数在指数和固定相位上都有稳步增加 (图 2B)。由于两种区域性都是以相同的光密度 (?...

Watch this Scientific Journal Video about Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis at JoVE.com

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

在这里, 我们描述了一个获得脂滴指数 (LD 指数) 的协议, 以研究在高通量实验中培养的细胞中甘油三酯的动力学。LD 指数法是一种使用 BODIPY 493/503 的简便可靠的方法。这种检测不需要 dispendious 脂提取或显微分析。

真菌病原体液体荧光恢复法测定脂质指数黑穗病玉米

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in ...

lipid-index-determination-liquid-fluorescence-recovery-fungal

Research

JoVE Journal

Biochemistry

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

7.5K 次观看.  Instituto Politécnico Nacional. 在这里, 我们描述了一个获得脂滴指数 (LD 指数) 的协议, 以研究在高通量实验中培养的细胞中甘油三酯的动力学。LD 指数法是一种使用 BODIPY 493/503 的简便可靠的方法。这种检测不需要 dispendious 脂提取或显微分析。

视频: Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

测定蓝细菌中的糖原含量

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

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

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

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 ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

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

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

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 ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

通过质量光度法快速测定抗体抗原亲和力

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

酶的结晶和结构确定:多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

通过代谢标记和薄层色谱分析 糖浆 中中性脂质合成

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...