Name: detection of protease activity by fluorescent peptide zymography
Uploaded: 2019-01-20T14:45:00
Description: Watch this Scientific Journal Video about Detection of Protease Activity by Fluorescent Peptide Zymography at JoVE.com

由俄亥俄州立大学工程学院生物医学工程系和综合癌症中心----arthur g. james 肿瘤医院和 richard j. solove 研究所提供的资金。

1. 解决凝胶层的制备
<ol><li>根据表 1准备10% 的聚丙烯酰胺解析凝胶溶液。在浇注凝胶之前立即加入四甲基二胺 (temed) 和过硫酸铵 (aps), 因为它们的添加会引发聚合反应。</li>
	<li>用10% 的解析凝胶溶液填充一个空的1.5 毫米小凝胶盒 (5 毫升)。</li>
	<li>在聚丙烯酰胺凝胶顶部添加一层薄薄的异丙醇 (~ 500μl), 以产生水平凝胶并防止气泡。使用剩余的聚丙烯酰胺溶液来跟踪聚合反应的进展。?...

<ol>
	<li>Vandooren, J., Geurts, N., Martens, E., Vanden Steen, P. E., Opdenakker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymography+methods+for+visualizing+hydrolytic+enzymes.">Zymography methods for visualizing hydrolytic enzymes.</a> Nature Methods. 10 (3), 211-220 (2013).</li><li>Toth, M., Fridman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Gelatinases+(MMP-2+and+MMP-9)+by+Gelatin+Zymography.">Assessment of Gelatinases (MMP-2 and MMP-9) by Gelatin Zymography.</a> Methods in Molecular Medicine. 57, (2001).</li><li>Heussen, C., Dowdle, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+analysis+of+plasminogen+activators+in+polyacrylamide+gels+containing+sodium+dodecyl+sulfate+and+copolymerized+substrates.">Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates.</a> Analytical Biochemistry. 102 (1), 196-202 (1980).</li><li>Gogly, B., Groult, N., Hornebeck, W., Godeau, G., Pellat, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+zymography+as+a+sensitive+and+specific+technique+for+the+determination+of+subpicogram+levels+of+interstitial+collagenase.">Collagen zymography as a sensitive and specific technique for the determination of subpicogram levels of interstitial collagenase.</a> Analytical Biochemistry. 255 (2), 211-216 (1998).</li><li>Inanc, S., Keles, D., Oktay, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+collagen+zymography+approach+for+evaluating+the+collagenases+MMP-1,+MMP-8,+and+MMP-13.">An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.</a> BioTechniques. 63 (4), 174-180 (2017).</li><li>Perera, H. K. I. <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+Aspartic+Proteinase+Activities+Using+Gel+Zymography.">Detection of Aspartic Proteinase Activities Using Gel Zymography.</a> Zymography. , 43-52 (2017).</li><li>van Beurden, P. A. M. S. n. o. e. k. -., Vonden Hoff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymographic+techniques+for+the+analysis+of+matrix+metalloproteinases+and+their+inhibitors.">Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.</a> BioTechniques. 38 (1), 73-83 (2005).</li><li>Oliver, G. W., Stetler-Stevenson, W. G., Kleiner, D. E., Zymography, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymography,+Casein+Zymography,+and+Reverse+Zymography:+Activity+Assays+for+Proteases+and+their+Inhibitors.">Zymography, Casein Zymography, and Reverse Zymography: Activity Assays for Proteases and their Inhibitors.</a> Proteolytic Enzymes. Springer Lab Manual. , 63-76 (1999).</li><li>Deshmukh, A. A., Weist, J. L., Leight, 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=Detection+of+proteolytic+activity+by+covalent+tethering+of+fluorogenic+substrates+in+zymogram+gels.">Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels.</a> BioTechniques. 64 (5), 203-210 (2018).</li><li>Yasothornsrikul, S., Hook, V. Y. <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+proteolytic+activity+by+fluorescent+zymogram+in-gel+assays.">Detection of proteolytic activity by fluorescent zymogram in-gel assays.</a> BioTechniques. 28 (6), 1172-1173 (2000).</li><li>Leight, J. L., Alge, D. L., Maier, A. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+matrix+metalloproteinase+activity+in+3D+cellular+microenvironments+using+a+fluorogenic+peptide+substrate.">Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.</a> Biomaterials. 34 (30), 7344-7352 (2013).</li><li>Leight, J. L., Tokuda, E. Y., Jones, C. E., Lin, A. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+bioscaffolds+for+3D+culture+of+melanoma+cells+reveal+increased+MMP+activity+and+migration+with+BRAF+kinase+inhibition.">Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (17), 5366-5371 (2015).</li><li>Ren, Z., Chen, J., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymography+as+a+Research+Tool+in+the+Study+of+Matrix+Metalloproteinase+Inhibitors.">Zymography as a Research Tool in the Study of Matrix Metalloproteinase Inhibitors.</a> Methods in Molecular Biology. 1626, 79-102 (2017).</li><li>Nagase, H., Fields, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+matrix+metalloproteinase+specificity+studies+using+collagen+sequence-based+synthetic+peptide.">Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptide.</a> Biopolymers. 40 (4), 399-416 (1996).</li><li>Mucha, A., 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=Membrane+Type-1+Matrix+Metalloprotease+and+Stromelysin-3+Cleave+More+Efficiently+Synthetic+Substrates+Containing+Unusual+Amino+Acids+in+Their+P1&#8242;+Positions.">Membrane Type-1 Matrix Metalloprotease and Stromelysin-3 Cleave More Efficiently Synthetic Substrates Containing Unusual Amino Acids in Their P1&#8242; Positions.</a> Journal of Biological Chemistry. 273 (5), 2763-2768 (1998).</li><li>Thimon, V., Belghazi, M., Labas, V., Dacheux, J. -. L., Gatti, 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=One-+and+two-dimensional+SDS-PAGE+zymography+with+quenched+fluorogenic+substrates+provides+identification+of+biological+fluid+proteases+by+direct+mass+spectrometry.">One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry.</a> Analytical Biochemistry. 375 (2), 382-384 (2008).</li><li>Sun, X., Salih, E., Oppenheim, F. G., Helmerhorst, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+mass+spectrometric+characterization+of+proteases+and+inhibitors+in+human+saliva.">Activity-based mass spectrometric characterization of proteases and inhibitors in human saliva.</a> Proteomics. Clinical Applications. 3 (7), 810-820 (2009).</li><li>Yu, W., Woessner, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-Enhanced+Zymographic+Detection+of+Matrilysin+and+Collagenases.">Heparin-Enhanced Zymographic Detection of Matrilysin and Collagenases.</a> Analytical Biochemistry. 293 (1), 38-42 (2001).</li></ol>

目前的酶学技术依赖于将原生底物纳入聚丙烯酰胺凝胶来检测蛋白溶解。虽然这些技术已经得到广泛的使用, 他们仍然是有限的蛋白酶, 他们可以检测到的数量。在这里, 描述了一个协议, 其中荧光, 蛋白酶降解肽被纳入聚丙烯酰胺解析凝胶。使用 azidi-pga-马来酰亚胺链接器分子进行共价偶联, 可分离和检测比目前本地底物更广泛的蛋白酶。荧光肽的高度可调谐特性使研究人员能够设计出能够针对其?...

凝胶酶学是一种生物技术, 用于测量生物样本中的蛋白溶解活性, 如体液或细胞培养介质 1,2,3。样品通过嵌入可降解基板的聚丙烯酰胺凝胶, 通过电泳的分子量分离。常见的可降解底物包括明胶、酪蛋白、胶原蛋白和弹性蛋白, 这些物质已被用来测量基质金属蛋白酶 (mmp)-1、-2、-3、-7、-8、-9 和-11, 此外还有各种导管 1, 2,4 个,5,6,7.,8. 电泳后, 酶被重新修饰, 并允许降解凝胶内的蛋白质。在传统的凝胶酶学中, 凝胶被蛋白质染料 (如 Coomassie) 染色, 蛋白酶活性被检测为信号的损失,即深蓝色背景上的白色带 (蛋白质降解)。
在这里, 我们描述了一种替代的凝胶酶学方法的协议, 其中可降解的底物是一个短的, 荧光肽共价地纳入聚丙烯酰胺凝胶 (图 1)。与传统的凝胶酶学相比, 合成肽作为可降解的底物的替代, 可以检测到更广泛的蛋白酶, 而传统的凝胶酶学方法是与原生蛋白质9相比。荧光肽的共价连接可防止凝胶电泳过程中的肽扩散和迁移, 用以前的方法9,10观察到。此外, 使用氟基板可以直接检测蛋白酶活性, 而无需额外的染色和去染色步骤。该方法的总体目标是通过在合氧化酶凝胶中共价地加入氟生成肽来检测生物样品中的蛋白酶活性。

<table>
	<tbody>
		<tr>
			<td>1.5 mm Empty Gel Cassettes</td>
			<td>ThermoFisher Scientific</td>
			<td>NC2015</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mm, 10 well Empty Gel Cassette Combs</td>
			<td>ThermoFisher Scientific</td>
			<td>NC3510</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>10-010-049</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS Solution</td>
			<td>Ambion</td>
			<td>AM9820</td>
			<td></td>
		</tr>
		<tr>
			<td>3x Zymography Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610764</td>
			<td></td>
		</tr>
		<tr>
			<td>40% (w/v) Acrylamide/Bis (19:1)</td>
			<td>Ambion</td>
			<td>AM9022</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Tissue Culture Plates</td>
			<td>ThermoFisher Scientific</td>
			<td>087721B</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit (10 kDa MWCO)</td>
			<td>Sigma-Aldrich</td>
			<td>UFC201024</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammounium Persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Azido-PEG3-Maleimide Kit</td>
			<td>Click Chemistry Tools</td>
			<td>AZ107</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>BP510100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>BP231</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A416P</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>N N N&#39; N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standard</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Hypodermic Needles</td>
			<td>Fisher Scientific</td>
			<td>14-826</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Bottom Flask (100 mL)</td>
			<td>Fisher Scientific</td>
			<td>50-873-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Septum Rubber Stopper</td>
			<td>Fisher Scientific</td>
			<td>50-872-546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Slip Tip Syringe (1 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-823-434</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma hydrochlroide</td>
			<td>Sigma-Aldrich</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon 9410 Molecular Imager</td>
			<td>GE Amersham</td>
			<td>8149-30-9410</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of protease activity by fluorescent peptide zymography

该方法的目的是测量复杂生物样品的蛋白溶解活性。通过嵌入可降解底物的解析凝胶, 通过电泳对样品进行分子量分离。这种方法不同于传统的凝胶酶学, 因为淬火的荧光肽是共价地纳入解决凝胶, 而不是全长蛋白质, 如明胶或酪蛋白。使用荧光肽可以直接检测蛋白溶解活性, 而无需额外的染色步骤。生物样品中的酶将淬火的荧光肽裂开, 从而增加荧光。然后用标准的荧光凝胶扫描仪对凝胶中的荧光信号进行成像, 并使用密度测量进行量化。多肽作为可降解底物的使用极大地扩展了利用酶成像技术检测到的可能的蛋白酶。

使用此处描述的方法, 将两种荧光蛋白酶降解肽纳入聚丙烯酰胺凝胶中: ggpqg iwqk (peg) 2c(在整个文本和数字中简称 qgiw) 和 gpla cpMeOBzl wark(peg) 2c (在整个案文和数字中缩写为 lacw)。表示的部位。qgiw 是一个用于检测细胞胶原酶14的胶原蛋白-i 派生序列。lacw 是一个针对检测 mmp-14 和 mmp-1115 进行优化的序列。这些肽用 n-?...

Watch this Scientific Journal Video about Detection of Protease Activity by Fluorescent Peptide Zymography at JoVE.com

Detection of Protease Activity by Fluorescent Peptide Zymography

在这里, 我们提出了一个详细的协议, 修改酶图技术, 其中荧光肽被用作可降解的底物, 而不是原生蛋白质。生物样品在荧光肽酶谱中的电泳使检测到比以前的酶学技术更广泛的蛋白酶。

荧光肽酶谱法检测蛋白酶活性

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

目前的酶学技术检测出数量有限的蛋白酶。荧光肽酶学使用荧光肽作为可降解基质，从而能够测量更大范围的蛋白酶。这种方法的主要优点是模块化设计。荧光肽的序列决定了检测到哪些蛋白酶，荧光肽可以很容易地与不同序列的肽交换，即检测其他蛋白酶的能力。该技术有助于为肽在工程生物材料（如用于药物输送或组织工程应用）中作为可降解交链剂的用途提供信息。将这种肽加入这种技术可以识别导致生物材料降解的组织分泌蛋白酶。演示此技术对于可视化与其他酶成像技术相比独一无二的多层方法非常重要。展示这种技术将是，阿梅亚德什穆赫，一个研究生从我的实验室。首先，准备一个10%聚丙烯酰胺解凝胶溶液，如文本协议表之一所述。在浇注凝胶之前，立即加入TEMED和PS。然后，用分解凝胶溶液填充并空空 1.5 毫米迷你凝胶盒。在聚丙烯酰胺凝胶的顶部加入一层薄薄的异丙醇，以产生水平凝胶并防止气泡。使用剩余的聚丙烯酰胺溶液跟踪聚合反应的进度。当管中的聚丙烯酰胺完全凝固时，反应是完整的。当第一个分解析凝胶层聚合时，从20摄氏度的储存中取回Azido-PEG3-Maleimide试剂盒，使组件达到室温。然后，溶解小瓶2的组件在制造商推荐的DMSO体积。和涡流30秒，以确保液体是良好的混合。将小瓶中的内容转移到一个干净、干燥的 100 毫升圆形底瓶中，其中含有搅拌棒。立即插入一个橡胶隔膜塞，用隔膜，用注射器刺穿到烧瓶口。接下来，将两根 18 规格的注射器针头插入隔膜。将其中一根注射器针头连接到惰性油箱，让气体在圆底烧瓶中加注三分钟。然后，关闭惰性气体，将其从针头中分离出来。使用注射器将小瓶二的全部内容注入烧瓶中。取出针头和注射器。在搅拌时，让部件在室温下混合 30 分钟。在此之后，拆下橡胶隔膜塞，将内装物转移到干净的五毫升离心管中。一旦第一个分子凝胶层聚合，就从异丙醇层中倾泻而下。将一毫升去离子水放在凝胶上，然后倒出水冲洗凝胶。从80摄氏度的储存中检索硫醇功能化荧光肽。允许在室温下解冻。准备含有Azido-PEG3-Maleimide链接剂分子和荧光肽的10%解孔凝胶溶液，如文本协议表之一所述。在浇注凝胶之前，立即添加 TMED 和 APS。接下来，用肽溶液填充凝胶盒剩余部分的一半。在聚丙烯酰胺凝胶的顶部加入一层薄薄的异丙醇，以产生水平凝胶并防止气泡。使用剩余的聚丙烯酰胺溶液跟踪聚合反应的进度。反应完成后，倒出异丙醇层。将一毫升去离子水放在凝胶上，然后倒出水冲洗凝胶。如果不立即使用凝胶，请将它们浸入一个塑料盒中，在摄氏四度时装满 100 毫升 1x PBS。用铝箔包裹盒子，防止光漂白。首先，准备一个5%堆叠凝胶溶液，如表之一的文本协议中所述。在浇注凝胶之前，立即添加 TMED 和 APS。用堆叠凝胶溶液填充凝胶盒的剩余空部分。快速将 1.5 毫米凝胶梳插入堆叠凝胶层，确保井下没有气泡。使用剩余的聚丙烯酰胺溶液跟踪聚合反应的进度。当反应完成后，轻轻地从凝胶盒背面取下梳子和胶带。首先，将样品溶解在传统的酶学样品缓冲液中。向凝胶设备添加 400 毫升 os 1X 三糖烯 SDS 运行缓冲液。每井最多装载 35 微升样品。在4摄氏度下以120伏度运行样品一个半小时，或直到分子量标准表明感兴趣的蛋白酶在肽解析凝胶层内。在此之后，从塑料盒中取下凝胶。在温和搅拌下，在室温下将凝胶在室温下重新洗涤缓冲液中清洗三次。每次洗涤，持续10分钟。将凝胶转移到开发中的缓冲液中 15 分钟。然后用新鲜发育的缓冲液代替溶液，在37摄氏度的温和搅拌下孵育24小时。之后，使用荧光凝胶扫描仪成像器与适当的激励和发射过滤器来成像凝胶。在这项研究中，将荧光蛋白酶可降解肽纳入聚丙烯酰胺凝胶中。QGIW 是一种胶原蛋白，用于检测细胞合发生，而 LACW 是一个针对 MMP-14 和 MMP-11 的检测进行了优化的序列。荧光成像揭示了LACW肽凝胶中的多个波段，而在QGIW凝胶中只看到一个波段。与明胶酶学相比，LACW凝胶能够检测出更多的蛋白糖解带，这证明肽酶学能够检测比使用原生基质的传统方法在生物样品中存在的更广泛的蛋白酶。然后，肽酶造影细胞在含有GM6001（广谱MMP抑制剂）或E64（一般导管素抑制剂）的发育缓冲液中孵育。使用 GM6001 治疗 LACW 肽凝胶，可降低带状强度。而E64的治疗没有明显的效果。使用 GM6001 处理 QGIQ 肽凝胶可完全消融以前看到的带子。而 E64 则不起作用。明胶酶造影使用纯化、活性的MMP-9进行，将肽酶学的敏感性与当前金标准进行比较。LACW凝胶能够检测MMP-9的最小浓度。蛋白酶需要特定的条件才能重新自然并被激活。仔细准备缓冲液，以确保成分和 PH 正确。此方法可以辅之以现有的技术，以验证身份，并执行测量蛋白酶的进一步分析。这包括西方印迹、实时 PCR 和质谱。处理聚丙烯酰胺时要小心。非多边化溶液被认为是一种有效的神经毒素，在处理时应始终佩戴适当的个人防护设备。

Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

胰腺癌的荧光原位小鼠模型

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

科研

癌症研究

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

体外成像和荧光标记的GnRH类似物的药物靶向性的定量分析

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

淋巴结慢病毒北疆-转基因 Organoids 培养的大鼠结肠肿瘤的高灵敏度检测

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

长距离反向PCR对热线1的逆变活性的检测

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...