Name: quantitative immunoblotting of cell lines as a standard to validate immunofluorescence for quantifying biomarker proteins in routine tissue samples
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples at JoVE.co

这项工作的部分资金来自弗雷德里克·班廷和查尔斯最佳加拿大研究生奖学金 (上午)。

使用初级人体组织样本的申请获得了女王大学卫生科学和附属教学医院研究伦理委员会 (hsreb) 的批准。
1. 建立细胞线组织微阵列 (tma)
<ol><li>收获和清洗细胞。 注: 该议定书已在各种已建立的永生细胞系 (例如hela、jurkat、rch-acv) 上进行了测试。<ol>
<li>对于粘附细胞, 一旦细胞达到约 80%的融合, 就会收获大约 1.3 x 10 7 细胞。使用适合该细胞系的试剂分离细胞。<b...

<ol>
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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=Validation+of+tumor+protein+marker+quantification+by+two+independent+automated+immunofluorescence+image+analysis+platforms.">Validation of tumor protein marker quantification by two independent automated immunofluorescence image analysis platforms.</a> Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc. 29 (10), 1143-1154 (2016).</li><li>Toki, M. I., Cecchi, F., Hembrough, T., Syrigos, K. N., Rimm, D. 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I., 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=First+application+of+the+Automated+QUantitative+Analysis+(AQUA)+technique+to+quantify+PTEN+protein+expression+in+ovarian+cancer:+A+correlative+study+of+NCIC+CTG+OV.16.">First application of the Automated QUantitative Analysis (AQUA) technique to quantify PTEN protein expression in ovarian cancer: A correlative study of NCIC CTG OV.16.</a> Gynecologic Oncology. 140 (3), 486-493 (2016).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+embedding+tissue+samples+for+sectioning.">Paraffin embedding tissue samples for sectioning.</a> CSH protocols. , (2008).</li><li>Fedor, H. L., Marzo, A. 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我们已经描述了一种方法, 利用定量免疫印迹 (ib) 来证明免疫荧光 (if) 的作用, 以确定在 ffpe 组织样本中的目标蛋白的相对丰度。目前的蛋白质定量方法受到其分类性质的限制, 如显色性 ihc2,3, 或需要对样品进行同质化, 防止对样本结构和细胞群的调查, 如与 ib 和质谱法8,9,10,<s...

在许多临床领域都需要量化福尔马林固定、石蜡包埋 (ffpe) 组织活检样本中的蛋白质。例如, 使用常规活检标本中生物标志物蛋白的定量来阐明预后, 并为癌症患者的治疗提供信息1。然而, 目前的方法通常是主观的, 缺乏验证。
免疫组织化学 (ihc) 通常用于病理实验室, 通常依赖于针对目标蛋白的初级抗体和与酶标签 (如辣根过氧化物酶 2) 结合的二级抗体.常规 ihc 是敏感的, 可以利用微小样本, 并保持组织样本的形态完整性, 从而允许评估其相关的组织学背景下的蛋白质表达。然而, 由于 ihc 产生的显色信号是减法的, 它的动态范围相对狭窄, 多路复用 2,3,4的潜力有限。矩阵辅助激光去光/电离质谱成像 (aldi-msi) 保留了形态完整性。然而, 这种开发的技术与适度的形态分辨率相关, 需要进行大量的校准和归一化, 从而损害了其常规临床使用的可行性 5, 6,7.在组织样本中量化蛋白质的替代技术包括免疫印迹8、质谱法 9、10、11和酶联免疫吸附法 (elisa)12, 每个其中开始于样品组织的均质裂解物。主要组织样本是异质性的, 因为它们包含多种细胞类型。因此, 需要对样品进行同质化的技术不允许对特定细胞群 (如癌细胞) 中的蛋白质进行定量。
与 ihc 一样, if 适用于小型 ffpe 样品, 并允许保留组织学完整性13。然而, 由于荧光信号的加性, if 适用于多种原生抗体和荧光标签的应用。因此, 感兴趣的蛋白质可以在使用其他抗体定义的特定细胞或细胞隔间 (例如, 细胞核与细胞质) 中相对量化。荧光信号也具有更大的动态范围13,14的优势。研究了if在ffpe 样品中的优越性、重现性和复用潜力。
在此, 我们描述了使用定量免疫印迹使用已建立的细胞系作为一个黄金标准, 以确定 if 的定量性质, 并结合计算机辅助图像分析, 以确定感兴趣的蛋白质的相对丰度ffpe 组织样本中的组织学切片。我们已经成功地应用了这种方法在多方法量化生物标志物蛋白在临床活检样本16,17,18.

<table border="0" cellpadding="0" cellspacing="0" style="width:675px;" width="673">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">697</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 42</td>
			<td style="width:213px;">Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">JeKo-1</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CRL-3006</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Jurkat</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">TIB-152</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">RCH-ACV</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 548</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Granta-519</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 342</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">REH</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 22</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Raji</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CCL-86</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">HeLa</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CCL-2</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Trypsin/EDTA solution</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:123px;">R001100</td>
			<td>For detaching adhesive cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fetal bovine serum (FBS)</td>
			<td style="width:116px;">Wisent Inc.</td>
			<td style="width:123px;">81150</td>
			<td>To neutralize trypsin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Neutral Buffered Formalin</td>
			<td style="width:116px;">Protocol</td>
			<td style="width:123px;">245-684</td>
			<td>For fixing cell pellets</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">UltraPure low melting point agarose</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:123px;">15517-022</td>
			<td>For casting cell pellets</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Mouse monoclonal anti-human Bcl-2 antibody, clone 124</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">cat#: M088729-2, RRID: 2064429</td>
			<td style="width:213px;">To detect Bcl-2 by immunoflourencence and immunoblot (lot#:&#160;00095786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Ventana Discovery XT</td>
			<td style="width:116px;">Roche</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">For automation of immunofluorescence staining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">EnVision+ System- HRP labelled polymer (anti-mouse)</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">K4000</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">EnVision+ System- HRP labelled polymer (anti-rabbit)</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">K4002</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Cyanine 5 tyramide reagent</td>
			<td style="width:116px;">Perkin Elmer</td>
			<td style="width:123px;">NEL745001KT</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Aperio ImageScope</td>
			<td style="width:116px;">Leica Biosystems</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">To view scanned slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">HALO image analysis software</td>
			<td style="width:116px;">Indica Labs</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">For quantification of immunofluorescence</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Protease inhibitors (Halt protease inhibitor cocktail, 100X)</td>
			<td style="width:116px;">Thermo Scientific</td>
			<td style="width:123px;">1862209</td>
			<td>To add to RIPA buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">EDT001</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NP-40</td>
			<td style="width:116px;">BDH Limited</td>
			<td style="width:123px;">56009</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Sodium deoxycholate</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:123px;">D6750</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glycerol</td>
			<td style="width:116px;">FisherBiotech</td>
			<td style="width:123px;">BP229</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bromophenol blue</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">BRO777</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dithiothreitol (DTT)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">161-0611</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bovine serum albumin (BSA)</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">ALB001</td>
			<td>For immunoblot washes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Protein ladder (Precision Plus Protein Dual Color Standards)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">161-0374</td>
			<td>For running protein gel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter paper (Extra thick blot paper)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">1703969</td>
			<td>For blotting transfer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Nitrocellulose membrane</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">162-0115</td>
			<td>For blotting transfer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Trans-blot SD semi-dry transfer cell</td>
			<td style="width:116px;">Bio-Rad</td>
			<td>1703940</td>
			<td>For semi-dry transfer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Skim milk powder (Nonfat dry milk)</td>
			<td style="width:116px;">Cell Signaling Technology</td>
			<td style="width:123px;">9999S</td>
			<td>For blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tween 20</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">TWN510</td>
			<td>For wash buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">GAPDH rabbit monoclonal antibody</td>
			<td style="width:116px;">Epitomics</td>
			<td style="width:123px;">2251-1</td>
			<td style="width:213px;">Primary antibody of control protein (lot#: YE101901C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Goat anti-mouse IgG (HRP conjugated) antibody</td>
			<td style="width:116px;">abcam</td>
			<td style="width:123px;">cat#: ab6789, RRID: AB_955439</td>
			<td style="width:213px;">Secondary antibody for immunoblot</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Goat anti-rabbit IgG (HRP conjugated) antibody</td>
			<td style="width:116px;">abcam</td>
			<td style="width:123px;">cat#: ab6721, RRID: AB_955447</td>
			<td style="width:213px;">Secondary antibody for immunoblot (lot#: GR3192725-5)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Clarity Western ECL substrates</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">1705060</td>
			<td style="width:213px;">For immunoblot signal detection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Amersham Imager 600</td>
			<td style="width:116px;">GE Healthcare Life Sciences</td>
			<td style="width:123px;">29083461</td>
			<td>For immunoblot signal detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">ImageJ software</td>
			<td style="width:116px;">Freeware, NIH</td>
			<td style="width:123px;">-</td>
			<td>For densitometry analysis</td>
		</tr>
	</tbody>
</table>

quantitative immunoblotting of cell lines as a standard to validate immunofluorescence for quantifying biomarker proteins in routine tissue samples

福尔马林固定、石蜡包埋 (ffpe) 组织样品中感兴趣的蛋白质的定量在临床和研究应用中具有重要意义。定量的最佳方法是准确的, 具有广泛的线性动态范围, 并保持样品的结构完整性, 以便识别单个细胞类型。目前的方法, 如免疫组织化学 (ihc), 质谱和免疫印迹每个不符合这些规定, 由于其分类性质或需要使样品同质化。作为另一种方法, 我们建议使用免疫荧光 (if) 和图像分析来确定 ffpe 组织中感兴趣的蛋白质的相对丰度。在此基础上, 我们证明了该方法易于优化, 产生了广泛的动态范围, 并与定量免疫印迹的黄金标准相比是线性可量化的。此外, 这种方法允许保持样品的结构完整性, 并允许区分各种细胞类型, 这在诊断应用中可能是至关重要的。总体而言, 这是一种用于 ffpe 样品中蛋白质相对定量的可靠方法, 可轻松适应临床或研究需求。

该方案用于确定 if 在构建 ffpe 组织块的细胞系中确定抗凋亡蛋白 bcl-2 的相对数量的能力。在癌细胞中选择性地量化 bcl-2 可以阐明致癌机制, 并可用于病理诊断和为临床管理决策提供信息 24.更具体地说, bcl-2 在适当的 b 淋巴细胞发育中起着重要作用, 其表达通常在淋巴瘤25、26、27的背?...

Watch this Scientific Journal Video about Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples at JoVE.co

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

我们描述了使用定量免疫印迹来验证免疫荧光组织学结合图像分析作为一种手段, 定量的蛋白质感兴趣的福尔马林固定, 石蜡嵌入 (ffpe) 组织样本。我们的研究结果证明了免疫荧光组织学在确定常规活检样本中生物标志物蛋白的相对数量方面的作用。

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

这种方法可以通过从常规处理活检材料中量化生物标志物蛋白来帮助回答癌症病理学领域关键问题。该技术与传统免疫基础化学相关的主要优点是客观、动态范围广，并允许特定细胞类型的蛋白质定量化。某些所谓的生物标志蛋白的存在或缺失，或者更具体地而言，癌症活检样本中这些蛋白质的相对丰度对于对特定种类的癌症进行特定的病理诊断非常有用。但它也可以在预测癌症治疗的反应，这就是为什么这种技术是相关的诊断和治疗癌症患者。该协议主要涉及如何在不朽细胞系中使用蛋白质定量来验证免疫荧光分析的定量性质。但需要注意的是，免疫荧光可以很容易地被纳入多路复用方法，并应用于初级活检样本。协助和演示这一程序将由技术员李·布德劳和运营总监沙克尔·维克担任。两者都来自女王分子病理学实验室。首先，将先前收获的细胞在225 G的50毫升锥形管中离心5分钟。去光上一提液，将细胞颗粒重新在10毫升PBS中。然后，将重新暂停的电池在225 G下离心5分钟。用 10 毫升 10%NBF 悬浮细胞颗粒。然后在室温下在摇杆上孵育细胞，在24个RM下过夜。固定细胞后，将细胞在225 G下离心5分钟。然后去除上一除水，在500微升PBS中重新暂停细胞。将细胞转移到1.5毫升微离心管中，通过离心使细胞产生颗粒。然后，吸上一道。将大约500微升的1%agarose溶液添加到每个含有细胞的管中。然后，将混合物上下移液混合。在阿加罗斯细胞溶液硬化后，在管中加入10%NBF的毫升。稍后，从样本中删除 NBF。使用剃须刀刀片卸下电池插头，然后将插头放入塑料纸巾盒中。使用自动组织处理器隔夜处理样品。然后，使用标准组织学方法将样品嵌入石蜡中。使用组织阵列仪，从石蜡块中收集 6 毫米核心，并将其插入空石蜡块中。在此之后，使用微原子准备新创建的细胞系TMA的两个组织学部分。然后安装组织学幻灯片上的部分，并去仿一些。首先，将细胞系TMA的两个幻灯片加载到自动染色系统中。用最佳原抗体稀释一侧染色，将另一侧作为阴性控制幻灯片。对两张幻灯片，应用核反面和二级抗体。染色过程后，加入酪氨酸信号放大试剂。在此之后，使用适当的激发和检测波长扫描使用使用的荧光团的免疫污化幻灯片。使用图像分析软件识别感兴趣的细胞隔间，无论是核还是细胞质，并量化每条线的均度荧光强度或MFI。为了确定哪个细胞系具有最丰富的靶蛋白，等分培养的细胞将分解成微离心管。然后添加 2.5 微升的 6x 层缓冲液，并加入足够的 RIPA 裂解缓冲液，使总体积达到 15 微升。接下来，用蛋白质梯和先前准备的样品加载 SDS 页面凝胶。在此之后，将空孔中加载空孔。运行凝胶，直到蓝色染料到达板的底部。执行半干蛋白转移后，使用剃刀刀片水平切割膜，将感兴趣的蛋白质与控制蛋白分离。根据制造商的说明进行阻断和抗体孵化。然后将膜条放在一个透明塑料袋中。使用 P1000 移液器用 ECL 混合物覆盖膜。然后密封袋子，在黑暗中室温下孵育膜一到两分钟。接下来，将包含膜的塑料袋放在数字成像平台中。使用化学发光和色度标记检测来捕获膜的各种曝光。使用图像分析软件或视觉观察，确定哪个细胞系表达最靶向蛋白质。免疫后，该细胞系的连续稀释，使用图像分析软件打开曝光图像。使用矩形选择工具选择凝胶的第一条车道。然后去分析，凝胶，并选择第一车道。通过将矩形选择工具移动到下一个车道来重复此过程，然后去分析、凝胶并选择下一个车道。接下来，去分析，凝胶，和绘图车道。使用直线工具在每个峰的基座上绘制线条，以消除背景噪音。然后使用魔杖工具选择每个峰值，然后从结果窗口获取每个峰值的带强度。使用密度测定输出，创建带强度的散射图，与每个主要抗体的总蛋白质量。然后，使用最佳拟合和视觉观察线来确定每个抗体的线性动态范围的位置。选择在线性范围内产生带强度的蛋白质浓度，并执行所有细胞系的免疫布洛特与该蛋白质浓度，如前面证明。接下来，对数字扫描进行密度测定，选择在每个抗体之前确定的线性范围内产生信号的曝光。在此之后，计算每个细胞系的目标蛋白带强度与加载控制带强度的比率。最后，使用统计软件在从 IF 染色图像分析中获得的值与从免疫印迹中获得的值之间执行 Pearson 相关测试。该协议用于确认 IF 确定抗凋亡蛋白 Bcl-2 的相对数量的能力。用免疫功能染色剂在人类扁桃体组织上测试了原抗体的稀释。对于这个实验，1到50的稀释被确定为最优的稀释，产生一个强烈的信号，几乎没有背景噪声。这种稀释用于通过免疫荧光染色细胞系TMA，图像分析用于量化归因于Bfl-2的细胞质Cy5信号。根据所有测试细胞系的免疫布洛特，Granta-519具有最大的 Bcl-2 丰度。然后，利用格兰塔-519 lysate的序列稀释的免疫布洛特来查找Bfl-2的线性动态范围，并控制蛋白质GAPDH。在此测定中，Bcl-2 的动态范围范围从近 0 的波段强度到 7500 个单位。GAPDH 的动态范围范围从 3000 到 6500 个单位。在线性范围内的暴露下重复免疫布洛特。并计算了每个细胞系的Bfl-2与GAPDH的比率。Pearson 相关测试表明，免疫印迹的强度比与定量 IF 的强度读数呈强而正相关。在尝试此过程时，重要的是要记住在多个时间点中暴露最终膜，以便找到所有波段都在其各自线性范围内的最佳曝光。验证 IF 协议的定量性质后，可以在常规处理的组织样本中修改为多路复用 IF，以便回答临床问题，如目标蛋白的表达位置。

Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

A Quantitative Fitness Analysis Workflow

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

测量去泛素酶的活性细胞系和组织样品的方法

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...