Name: using microtiter dish radiolabeling for multiple in vivo measurements of escherichia coli (p)ppgpp followed by thin layer chromatography
Uploaded: 2019-06-04T14:00:00
Description: Watch this Scientific Journal Video about Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography at JoVE.com

这项研究得到了美国国家卫生研究院尤尼斯·肯尼迪·施莱佛国家儿童健康与人类发展研究所(Eunice Kennedy Shriver)内部研究计划的支持。

1. 媒体准备
<ol><li>
对于 MOPS (3-(N-morpholino) 丙烷硫酸) 介质25,使用 1/10 体积的 10x MOPS 盐、1/100 体积的 100x 微量营养素溶液、3 mM 磷酸钠用于过夜培养物或 0.2 mM 磷酸钠进行 32 P 均匀标记, 0.2% 葡萄糖和 1 μg/mL 三明(维生素 B1)。如果需要,在40μg/mL下加入氨基酸。
	<ol>
<li>对于 MOPS 盐,使用 400 mM MOPS、40 mM Tricine、0.1 mM FeSO 4、95 mM NH4Cl、2.6 mM K2SO4、...

<ol>
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C., Murakami, K. S., Tenson, T., Gerdes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+functional+insights+into+the+role+of+(p)ppGpp+in+bacterial+physiology.">Recent functional insights into the role of (p)ppGpp in bacterial physiology.</a> Nature Reviews Microbiology. 13 (5), 298-309 (2015).</li><li>Arenz, 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+stringent+factor+RelA+adopts+an+open+conformation+on+the+ribosome+to+stimulate+ppGpp+synthesis.">The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis.</a> Nucleic Acids Research. 44 (13), 6471-6481 (2016).</li><li>Loveland, A. 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=Ribosome&#8226;RelA+structures+reveal+the+mechanism+of+stringent+response+activation.">Ribosome&#8226;RelA structures reveal the mechanism of stringent response activation.</a> eLife. 5, e17029 (2016).</li><li>Brown, A., Fern&#225;ndez, I. S., Gordiyenko, Y., Ramakrishnan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome-dependent+activation+of+stringent+control.">Ribosome-dependent activation of stringent control.</a> Nature. 534 (7606), 277-280 (2016).</li><li>Battesti, A., Bouveret, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyl+carrier+protein/SpoT+interaction,+the+switch+linking+SpoT-dependent+stress+response+to+fatty+acid+metabolism.">Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism.</a> Molecular Microbiology. 62 (4), 1048-1063 (2006).</li><li>Lee, J. -. W., Park, Y. -. H., Seok, Y. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rsd+balances+(p)ppGpp+level+by+stimulating+the+hydrolase+activity+of+SpoT+during+carbon+source+downshift+in+Escherichia+coli.">Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (29), E6845-E6854 (2018).</li><li>Cashel, M. <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+(p)ppGpp+Accumulation+Patterns+in+Escherichia+coli+mutants.">Detection of (p)ppGpp Accumulation Patterns in Escherichia coli mutants.</a> Molecular Microbiology Techniques. 3, 341-356 (1994).</li><li>Fern&#225;ndez-Coll, L., Cashel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+SpoT+Hydrolase,+SpoT+Synthetase,+and+RelA+Synthetase+to+Carbon+Source+Diauxic+Growth+Transitions+in+Escherichia+coli.">Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli.</a> Frontiers in Microbiology. , (2018).</li><li>Varik, V., Oliveira, S. R. A., Hauryliuk, V., Tenson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC-based+quantification+of+bacterial+housekeeping+nucleotides+and+alarmone+messengers+ppGpp+and+pppGpp.">HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp.</a> Scientific Reports. 7 (1), 11022 (2017).</li><li>Rhee, H. -. 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=Selective+Fluorescent+Chemosensor+for+the+Bacterial+Alarmone+(p)ppGpp.">Selective Fluorescent Chemosensor for the Bacterial Alarmone (p)ppGpp.</a> J. Am. Chem. Soc. 130 (3), 784-785 (2008).</li><li>Wang, J., Chen, W., Liu, X., Wesdemiotis, C., Pang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mononuclear+Zinc+Complex+for+Selective+Detection+of+Diphosphate+via+Fluorescence+ESIPT+Turn-On.">A Mononuclear Zinc Complex for Selective Detection of Diphosphate via Fluorescence ESIPT Turn-On.</a> Journal of Materials Chemistry B. 2 (21), 3349-3354 (2014).</li><li>Pokhilko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+nutrient+limitation+in+growing+E.+coli:+a+mathematical+model+of+a+ppGpp-based+biosensor.">Monitoring of nutrient limitation in growing E. coli: a mathematical model of a ppGpp-based biosensor.</a> BMC Systems Biology. 11 (1), 106 (2017).</li><li>Funabashi, H., Mie, M., Yanagida, Y., Kobatake, E., Aizawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+monitoring+of+cellular+physiological+status+depending+on+the+accumulation+of+ppGpp.">Fluorescent monitoring of cellular physiological status depending on the accumulation of ppGpp.</a> Biotechnology Letters. 24 (4), 269-273 (2002).</li><li>Neidhardt, F. C., Bloch, P. L., Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+medium+for+enterobacteria.">Culture medium for enterobacteria.</a> Journal of Bacteriology. 119 (3), 736-747 (1974).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Peselis, A., Serganov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ykkC+riboswitches+employ+an+add-on+helix+to+adjust+specificity+for+polyanionic+ligands.">ykkC riboswitches employ an add-on helix to adjust specificity for polyanionic ligands.</a> Nature Chemical Biology. 14 (9), 887-894 (2018).</li><li>Oki, T., Yoshimoto, A., Sato, S., Takamatsu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purine+nucleotide+pyrophosphotransferase+from+Streptomyces+morookaensis,+capable+of+synthesizing+pppApp+and+pppGpp.">Purine nucleotide pyrophosphotransferase from Streptomyces morookaensis, capable of synthesizing pppApp and pppGpp.</a> Biochimica et Biophysica Acta (BBA) - Enzymology. 410 (2), 262-272 (1975).</li><li>Travers, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppApp+alters+transcriptional+selectivity+of+Escherichia+coli+RNA+polymerase.">ppApp alters transcriptional selectivity of Escherichia coli RNA polymerase.</a> FEBS Letters. 94 (2), 345-348 (1978).</li><li>Bruhn-Olszewska, 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=Structure-function+comparisons+of+(p)ppApp+vs+(p)ppGpp+for+Escherichia+coli+RNA+polymerase+binding+sites+and+for+rrnB+P1+promoter+regulatory+responses+in+vitro.">Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro.</a> Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1861 (8), 731-742 (2018).</li></ol>

实现细胞近乎均匀的标签是该协议的关键步骤。因此,使用定义的介质(如 MOPS 或 Tris 介质)对于允许携带磷酸盐浓度和特定活动的变化至关重要。不能使用磷酸盐缓冲介质,如 M9 或介质 A。大多数未定义的介质含有可变量的磷酸盐,如LB、胰锥酮和卡萨米诺酸。磷酸盐同位素33P是一种较弱的发射体,可以替代32 P。 然而,32 P的更高能量排放大大提高了检测灵敏度。必须强?...

(p)ppGpp第二信使是一个全球调节器,调节大量基因的表达,包括用于合成核糖体和氨基酸1、2的基因。虽然最初发现在大肠杆菌3,(p)ppGpp可以发现在革兰氏阳性和革兰氏阴性细菌,以及在植物叶绿体4,5。对于大肠杆菌和其他革兰氏阴性细菌,(p)ppGpp在6、7、8两个不同位点与RNA聚合酶直接相互作用。在革兰氏阳性,(p)ppGpp抑制GTP丰度,这是由CodY,一种GTP结合蛋白与基因特异性DNA识别主题,导致调控9,10。(p)ppGpp积累,以回应饥饿的不同营养和压力条件,导致缓慢生长和调整基因表达,使适应压力11,12。
ppGpp累积的净量反映了合成酶和水酶活动之间的平衡。在大肠杆菌RelA是一种强合成酶和SpoT是双功能,具有强水酶和弱合成酶,其中每一种可能以不同的方式调节在应力依赖的方式。强RelA合成酶被激活时,通币指定的带电tRNA绑定到核糖体A位点时,它未能跟上蛋白质合成13,14,15的需求。弱孢子(p)ppGpp合成酶被激活,而强(p)ppGpp水解酶被抑制,以响应其他应力条件并通过其他机制。在某些情况下,ACP或Rsd等蛋白质可以结合到SpoT,这也改变了水解和合成16、17之间的平衡。在革兰氏阳性物中,合成和水解反映了单一RelA SpoT同源(RSH)蛋白之间更复杂的平衡,具有很强的合成和水解活性,以及较小的水酶和/或合成酶12。
(p)ppGpp核苷酸首次被发现为不寻常的32P标记点,出现在薄层色谱图(TLC)的自动放射图上,在氨基酸饥饿3引起的严格反应。更详细的标签协议已经审查18。此处描述的协议(图1)是对这些协议的修改,允许监测微小板上多个样品的生长。这有利于对(p)ppGpp丰度变化的多重生物和技术估计,最初是为研究二代移位19而开发的。带有32P 和 TLC 检测的 (p) ppGpp 标签也允许测量 (p) ppGpp 降解率。已开发替代方法来确定(p)ppGpp水平,如质谱、HPLC20、荧光化学传感器21、22和GFP基因融合到受ppGpp23影响的启动子。24.荧光化学传感器目前用途有限,因为结合ppGpp后光谱偏移小,以及ppGpp和ppGpp21之间的问题。该方法在体外检测(p)ppGpp是有效的,但在细胞提取物中却不能。涉及HPLC的方法已经改进了20个,但需要昂贵的设备,并且不能很好地适应高通通。最后,GFP融合可以估计ppGpp依赖激活或抑制,但不测量ppGpp本身。虽然每种替代方法都是有价值的,但它们需要昂贵的设备或大量的动手时间,否则它们不能接受多种动力学采样和后续处理。通过本文所述的方法,96 个样品可在大约 20 分钟(每板 18 个样本)内应用于 6 个 TLC 板,TLC 开发可在几个小时内解决,在几个小时或一夜之间获得定量数据,具体取决于标签强度。

<table>
	<tbody>
		<tr>
			<td>(NH4)6(MO7)24</td>
			<td>Fisher Scientifics</td>
			<td>A-674</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoradiography film</td>
			<td>Denville scientific inc.</td>
			<td>E3218</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>J.T.Baker</td>
			<td>1-1309</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>RPI</td>
			<td>C61000-25.0</td>
			<td></td>
		</tr>
		<tr>
			<td>CoCl2</td>
			<td>Fisher Scientifics</td>
			<td>C-371</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>J.T.Baker</td>
			<td>1843</td>
			<td></td>
		</tr>
		<tr>
			<td>FeSO4</td>
			<td>Fisher Scientifics</td>
			<td>I-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Fisher Biotech</td>
			<td>BP1215-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Macron</td>
			<td>4912-12</td>
			<td></td>
		</tr>
		<tr>
			<td>H3BO4</td>
			<td>Macron</td>
			<td>2549-04</td>
			<td></td>
		</tr>
		<tr>
			<td>H3PO4</td>
			<td>J.T.Baker</td>
			<td>0260-02</td>
			<td></td>
		</tr>
		<tr>
			<td>K2SO4</td>
			<td>Sigma</td>
			<td>P9458-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Biotech</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>Sigma</td>
			<td>V-6504</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher Scientifics</td>
			<td>FL-06-0303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader Synergy HT</td>
			<td>Biotek</td>
			<td>Synergy HT</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma</td>
			<td>M-9522</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Mallinckrodt</td>
			<td>7892</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>J.T.Baker</td>
			<td>3624-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Mallinckrodt</td>
			<td>7917</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma</td>
			<td>A0171-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>P-32 radionuclide, orthophosphoric acid in 1 mL water (5 mCi)</td>
			<td>Perkin Elmer</td>
			<td>NEX053005MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage phosphor screen</td>
			<td>Kodak</td>
			<td>So230</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Sigma</td>
			<td>T-4625</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC PEI Cellulose F</td>
			<td>Merk-Millipore</td>
			<td>1.05579.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine</td>
			<td>RPI</td>
			<td>T2400-500.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon 9400 imager</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Fisher Scientifics</td>
			<td>Z-68</td>
			<td></td>
		</tr>
	</tbody>
</table>

using microtiter dish radiolabeling for multiple in vivo measurements of escherichia coli (p)ppgpp followed by thin layer chromatography

(p)ppGpp核苷酸作为细菌的全球调节器,对各种物理和营养压力作出反应。它有一个快速的开始,在几秒钟内,这导致积累的水平接近或超过的GTP池的水平。应力反转场合迅速消失(p)ppGpp,通常半寿命不到一分钟。(p)ppGpp的存在导致细胞基因表达和代谢的改变,以对抗压力的破坏性影响。革兰氏阴性和革兰氏阳性细菌有不同的反应机制,但都取决于(p)ppGpp浓度。在任何情况下,都需要同时监测许多放射性标记细菌培养物的时间间隔,在临界应力过渡期间,时间间隔可能从 10 秒到数小时不等。此协议解决了这一技术难题。该方法利用温度和摇摇控制微酸盐培养箱,允许平行监测生长(吸收)和均匀磷酸盐放射性标记培养物的快速采样,通过PEI纤维素上的薄层色谱。分析的多重技术和生物复制需要少量样品。复杂的增长过渡,如二次增长和快速(p)ppGpp周转率可以通过这种方法进行定量评估。

在大肠杆菌K-12菌株中,添加血管素引起异于明的内源性饥饿,导致5分3秒后ppGpp水平增加。在MOPS中生长的含有除ILV以外的所有氨基酸的细胞,如图1所示,标有32P。 一旦贴上标签,加入6 μL的10毫克/mL-Valine(100微克/mL最终浓度),以产生无盐饥饿。样品在添加静脉曲明后0和5分钟进行。5分钟后(图3A),ppGpp和pppGpp水平增加了2倍和2.5倍。作为...

Watch this Scientific Journal Video about Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography at JoVE.com

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

微酸度皿中放射性标记细菌培养物的生长有助于高通量采样,从而允许对核苷酸池丰度进行多种技术和生物复制检测,包括 (p) ppGpp。可以监测生理压力来源引起的生长转变以及压力恢复的影响。

使用微蒂特萝卜无线电标签进行多维欧大肠杆菌测量 (p) ppGpp,然后是薄层色谱

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...

该方法测量不同增长条件和压力情况下全球调节器 ppGpp 的水平。PpGpp 可以在克阳性和阴性细菌以及叶绿体中找到。PpGpp 在几秒钟内快速启动，一旦产生应力，即导致快速的第二次关系。同时，它有一个短的半寿命，长达30秒。因此，需要以时间间隔监控许多区域性，这些区域性的时间间隔可能从 10 秒到小时不同，包括压力过渡。我们还在微刺激器培养皿中对细菌培养进行放射性标记，从而促进高通量采样，从而允许进行多种技术和生物复制。处理放射性物质时，请务必遵循适当的安全措施。若要开始此过程，请准备文本协议中概述的所有媒体。在 MOPS 介质中生长带三毫摩尔磷酸钠的隔夜培养。加入550微升的MOS介质，在24孔板的井中连续增0.2微摩尔磷酸钠载体和30微升每个细菌 inculum。这将导致 OD600 为 0.1 的初始孵化。将新介质添加到无菌控制中。将板在 37 摄氏度的摇晃培养箱中，在 900 RPM 下摇晃 30 分钟。在缺少 p32 的介质中，使用板读卡器在相同条件下孵育时，可以使用具有复制板的板读取器进行实时监控生长。接下来，在板的每个井中加入100个p32磷酸盐的微曲线。在摇动的培养箱中生长培养培养物，使培养成一到两倍，使外部标签与细胞间核苷酸池基本平衡。为了正确可视化，此视频中未使用任何活动材质。在实际实验中，需要适当的屏蔽，以及使用测量仪持续监测污染情况。首先，使用适合所研究的压力排序的方法诱导应力。接下来，将每个标记细胞样品的20微升添加到单独的0.2毫升PCR管中，该管含有20微升的冰冷六摩尔甲酸。立即将样品放在干冰上。通过三个循环的冷冻和解冻提高细胞提取效率。在发现PEI纤维素薄层色谱图之前，以最大速度将样品离心一分钟，以颗粒细胞碎片。首先，设置一个20至20厘米的PEI纤维素TLC板。用剪刀去除前五厘米。并使用一支软铅笔将原点线从边缘标记一厘米。将每个样品的五微升液滴涂抹到PEI表面。在该点干燥之前，将板转移到含有1.5摩尔单钾磷酸盐层的罐中，该层较浅，不能接触原点样品点。用气密密封盖住油箱，让液体升到 15 厘米修剪的薄片的顶部。之后，取出完全开发的色谱，在室温下空气干燥。将含有自由 P32 的色谱图的顶部切割并丢弃为放射性废物。使用荧光屏在一夜之间曝光音频放射片。第二天，开发薄膜，并使用荧光粉来捕获和量化荧光粉的绿色信号。然后，使用 ImageJ 软件对放射性点进行定量化。在引发压力或饥饿的时间足以允许 ppGpp 积累后，使用适当的方法来扭转压力。每 20 到 30 秒采集一次样品，最多两分钟，然后像前面描述的那样处理样品。一旦开发TLC，并在时间过程中获得ppGpp的级别，在半历史图与时间上绘制剩余ppGpp内容。在这项研究中，在含有除 ILV 以外的所有氨基酸的 MOPS 中生长的细胞都标有 P32。一旦标记，L瓦林被添加产生二氧核素饥饿。五分钟后，瓜诺辛的四磷和五磷酸盐含量增加了两岁半。对无细胞标记样品进行负对，用于检测P32源中未为正交磷酸盐的可能化合物。抑制蛋白质合成的氯霉素可逆转二氯蔗素饥饿，从而减少带电的二氯蔗氨酸 tRNA 的消耗，进而恢复高充电与未充电 tRNA 的比率。强重按中移pp合成酶的激活被废除，这允许一种尺度的ppGpp降解不受残余合成的干扰。在这种情况下，ppGpp 衰减的半衰期约为 64 秒。重要的是让细胞生长几代人，以达到一个统一的标签之前，诱导任何压力或饥饿。由于ppGpp在细菌中广泛传播，因此可以应用于其他生物体。修改TLC开发方法可以正确分离所有调节性核苷酸，如ppApp。P52 是更好的发射同位素，因此使用适当的屏蔽和正确丢弃任何残留物非常重要。

Research

JoVE Journal

Biology

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

MALDI Sample Preparation: the Ultra Thin Layer Method

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

拟南芥极地甘油脂分析薄层色谱（TLC）再加上气相色谱（GLC）

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

对于电感受的制备快速协议<em&gt;大肠杆菌</em&gt;和<em&gt;霍乱弧菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...