Name: high throughput, absolute determination of the content of a selected protein at tissue levels using quantitative dot blot analysis (qdb)
Uploaded: 2018-08-21T13:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB at JoVE.com

这项工作得到了泰山学者建设工程 (G.T.)、山东优秀青年科学家奖 (ZR2016JL026 G.T.)、中国国家自然科学基金 (31771284、3167070448、81641108)、山东省自然科学基金会 (ZR2016JL026, 2017GSF18103), 山东省科技计划 (J14LE01 和 J15LK03), 烟台科技计划 (2015ZH083) 和滨州医科大学科研基金 (BY2013KYQD17, BY2013KYQD18),瑞典研究理事会赠款621-2011-4423 和2015-4870。 这项工作还由 "烟台双百人才计划" 和 "烟台高新区蓝海人才计划" 赞助。

所有动物程序均按照滨州医科大学动物使用指令进行, 并经滨州医科大学伦理审查委员会批准。
1. 样品准备
<ol><li>将50毫克小鼠组织成1.5 毫升管。添加200µL 裂解缓冲液 (50 毫米 Hepes, pH 7.4, 137 毫米氯化钠, 5 毫米 EDTA, 5 毫米 EGTA, 1 毫米氯化镁2, 10 毫米 Na2P2O7, 1% 海卫 X-100, 10% 甘油), 辅以蛋白酶和磷酸酶抑制剂 (100 毫米 NaF, 0.1 毫米 phenylmethylsulfonyl氟化物, 5 µg/毫升 pepstatin, 10 µg/毫升 leupeptin, 5 µg/毫升抑肽酶)。</li>
	<li>融汇在冰上用1分钟的匀质机组织。</li>
	<li>离心机为10分钟, 8 000 x 克, 4 摄氏度。</li>
	<li>将上清入新管 (1.5 毫升), 取出1µL 蛋白质浓度测定试剂盒。 注:所有常用的裂解缓冲剂用于西方印迹分析和斑点杂交分析, 可用于 QDB 分析。</li>
</ol>2. 测定抗体的特异性
<ol><li>准备样品裂解物 (20 µg) 在1.4 节, IgG 游离 BSA (20 µg), 标准蛋白 (600 pg), 为西方印迹分析。 注:无 IgG 的 BSA 被用作阴性对照, 标准蛋白作为一种阳性对照。用西方印迹分析法显示一条正确大小的波段, 证明了抗体的特异性。</li>
</ol>3. 定义 QDB 分析的线性范围
<ol><li>溶解标准蛋白与 ddH2o. 稀释蛋白质到1200页/µL 浓度。</li>
	<li>从1.4 至4µg/µL 中制备的样品中稀释浓缩蛋白。</li>
	<li>将 BSA 与 ddH2o 稀释蛋白质, 浓缩 1200 pg/µL, 4 µg/µL。</li>
	<li>在表 1和表 2的指导下, 编写了一系列标准蛋白和制备样品的稀释。</li>
	<li>混合10µL 稀释标准蛋白或稀释制备的样品和10µL 2x 加载缓冲器 (120mM 三盐酸 pH 6.8, 20% 甘油, 4% SDS, 0.2% 溴酚蓝, 200 毫米) 一起。</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>S1 (0 页/µl)</td>
			<td>S2 (33.3pg/µl)</td>
			<td>S3 (100 页/µl)</td>
			<td>S3 (300 页/µl)</td>
			<td>S5 (600 页/µl)</td>
			<td>S6 (1200 页/µl)</td>
		</tr>
<tr>
<td>标准蛋白 (1200 页/µl)</td>
			<td>0</td>
			<td>1.39</td>
			<td>4.17</td>
			<td>12。5</td>
			<td>25</td>
			<td>50</td>
		</tr>
<tr>
<td>无 IgG BSA (4 页/µl)</td>
			<td>50</td>
			<td>48.61</td>
			<td>45.83</td>
			<td>37。5</td>
			<td>25</td>
			<td>0</td>
		</tr>
<tr>
<td>总</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
		</tr>
</tbody></table>表1。标准蛋白质曲线的稀释方案。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>X1 (0µg/µl)</td>
			<td>X2 (0.25µg/µl)</td>
			<td>X3 (0.5µg/µl)</td>
			<td>X4 (1µg/µl)</td>
			<td>X5 (2µg/µl)</td>
			<td>X6 (4 µg/µl)</td>
		</tr>
<tr>
<td>样品 (4µg/µl)</td>
			<td>0</td>
			<td>3.125</td>
			<td>6.25</td>
			<td>12。5</td>
			<td>25</td>
			<td>50</td>
		</tr>
<tr>
<td>无 IgG BSA (4µg/µl)</td>
			<td>50</td>
			<td>46.875</td>
			<td>43.75</td>
			<td>37。5</td>
			<td>25</td>
			<td>0</td>
		</tr>
<tr>
<td>总</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
		</tr>
</tbody></table>表2。准备样品稀释方案
<ol start="6"><li>将混合物加热85摄氏度, 5 分钟。</li>
</ol>4. QDB 分析的过程
<ol><li>
示例应用程序
	<ol>
<li>支持 QDB 板, 避免板底接触台面表面。例如, 使用空吸管提示框作为支持。</li>
		<li>将样品的2µL 到 QDB 板的各个单元底部的膜中心。</li>
	</ol>
</li>
	<li>
烘干板材
	<ol>
<li>将加载的 QDB 板留在室温下 (RT), 或作为替代品, 在通风良好的空间中, 将所载的盘子在37摄氏度处放在15分钟。</li>
	</ol>
</li>
	<li>
阻挡板
	<ol>
<li>将 QDB 板浸在转移缓冲器中 (0.039 米甘氨酸, 0.048 米三, 0.37% SDS, 20% 甲醇), 轻轻摇动盘子十年代。</li>
		<li>用 TBST (三缓冲盐水, 0.1% 吐温 20) 轻轻冲洗 QDB 板三次, 然后在恒定晃动的 TBST 中将盘子洗净5分钟。</li>
		<li>在恒定晃动的情况下, 用阻塞缓冲器 (5% TBST (100 µL/井) 的 QDB 板堵住1小时。</li>
	</ol>
</li>
	<li>
原发性抗体孵化
	<ol>
<li>稀释在所选浓度 (从 1: 500 到 1:5 000) 的阻塞缓冲区中的主要抗体, 并增加100µL 到普通96井板的每一个人井。将 QDB 板插入96井板中, 并在连续晃动下, 在 RT 或隔夜4摄氏度的情况下, 将组合板孵化为2小时。</li>
		<li>或者, 将 QDB 板放在盒内, 如果用相同的抗体用于整片板, 则将盒内的阻塞缓冲器2至3毫米填满。在所选浓度下添加主抗体, 并在连续晃动下, 在 RT 或隔夜4摄氏度孵育2小时的平板。</li>
		<li>用 TBST 轻轻地冲洗盘子三次, 然后用 TBST 洗三次, 每次5分钟不停地摇晃。</li>
	</ol>
</li>
	<li>
次生抗体孵化
	<ol>
<li>稀释在所选浓度 (从1:1、000到1:50、000) 的阻塞缓冲器中的二次抗体, 整除100µL/井成96井板或4.4.2 节中所述的框中, 然后在加载的96井 pla 内孵化 QDB 板。te 或盒为1小时在 RT 下恒定的震动。</li>
		<li>用 TBST 轻轻冲洗 QDB 板3次, 然后将盘子洗净3次, 每 TBST 5 分钟, 在恒定的震动下。</li>
	</ol>
</li>
	<li>
量化
	<ol>
<li>按照制造商的指示, 制备增强化学发光基板 (ECL)。</li>
		<li>整除在96井板 (100 µL/井) 中, 将 QDB 板插入96井板内, 在恒定晃动下2分钟。</li>
		<li>从96井板上取下 QDB 板, 并简单地摇动以除去多余的液体。把盘子放在白色的微量滴定板上。</li>
		<li>打开微板块读取器, 然后在用户界面上选择 "带盖板", 然后再将组合板 (QDB 板 + 白版适配器) 放入微板块读卡器内进行量化。 注:确保使用白色, 非透明的微量滴定板作为适配器, 以避免任何干扰从板块。请务必选择 "盖板", 以避免干扰机器时, 组合板 (QDB 板 + 96 井板适配器) 放置在微板块读卡器。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Renart, J., Reiser, J., Stark, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera:+a+method+for+studying+antibody+specificity+and+antigen+structure.">Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.</a> Proc Natl Acad Sci U S A. 76 (7), 3116-3120 (1979).</li><li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Western+blotting":+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Anal Biochem. 112 (2), 195-203 (1981).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li><li>Uhlen, 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=A+proposal+for+validation+of+antibodies.">A proposal for validation of antibodies.</a> Nat Methods. 13 (10), 823-827 (2016).</li><li>Hawkes, R., Niday, E., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dot-immunobinding+assay+for+monoclonal+and+other+antibodies.">A dot-immunobinding assay for monoclonal and other antibodies.</a> Anal Biochem. 119 (1), 142-147 (1982).</li><li>Engvall, E., Perlmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+immunosorbent+assay+(ELISA).+Quantitative+assay+of+immunoglobulin.">Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin.</a> G. Immunochemistry. 8 (9), 871-874 (1971).</li><li>Engvall, E., Jonsson, K., Perlmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+immunosorbent+assay.+II.+Quantitative+assay+of+protein+antigen,+immunoglobulin+G,+by+means+of+enzyme-labelled+antigen+and+antibody-coated+tubes.">Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes.</a> Biochim Biophys Acta. 251 (3), 427-434 (1971).</li><li>Paweletz, C. P., 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=Reverse+phase+protein+microarrays+which+capture+disease+progression+show+activation+of+pro-survival+pathways+at+the+cancer+invasion+front.">Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.</a> Oncogene. 20 (16), 1981-1989 (2001).</li><li>Tibes, 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=Reverse+phase+protein+array:+validation+of+a+novel+proteomic+technology+and+utility+for+analysis+of+primary+leukemia+specimens+and+hematopoietic+stem+cells.">Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells.</a> Mol Cancer Ther. 5 (10), 2512-2521 (2006).</li><li>Solier, C., Langen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-based+proteomics+and+biomarker+research+-+current+status+and+limitations.">Antibody-based proteomics and biomarker research - current status and limitations.</a> Proteomics. 14 (6), 774-783 (2014).</li><li>Tian, G., 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=Quantitative+dot+blot+analysis+(QDB),+a+versatile+high+throughput+immunoblot+method.">Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.</a> Oncotarget. 8 (35), 58553-58562 (2017).</li></ol>

作者云云张、文峰和简狄张是 Zestern 生物技术的员工, 生产 QDB 板。云云和张先生宣布利益冲突, 简狄已申请专利。其他人声称没有相互竞争的利益。

除了 ELISA 之外, 目前所有现有的蛋白质分析免疫检测技术中, QDB 分析是在细胞和组织水平上以高通量格式实现特定蛋白质绝对含量的唯一方法。虽然用稳定的同位素标记标准, 质谱法能够实现对少数几种蛋白质的绝对量化, 但这种方法尚未设计出高吞吐量的性能。在本研究中, 我们展示了 QDB 分析的过程, 以达到绝对确定的 CAPG 蛋白水平的小鼠组织, 包括肾脏, 脾脏, 前列腺。CAPG 水平被发现在200到 360 pg/克之间的小鼠肾脏组织, 460 到 650 pg/g 在小鼠脾, 330 至 390 pg/g 在小鼠前列腺组织。
与 ELISA 相比, QDB 分析需要在常规实验室中进行最低限度的努力。首先, QDB 板是一种以硝化膜为基础, 消除酶联免疫吸附的涂层步骤。第二, QDB 分析只需要一个, 而不是两个特定的抗体在三明治 ELISA。第三, 抗硝化膜的高结合能力与 ELISA 板表面相比, 也允许膜经受住严格的洗涤步骤, 通常用于应用免疫印迹分析, 以减少背景干扰。这一特点是非常有用的分析复杂的裂解物制备的细胞和组织。相比之下, 由于 ELISA 板的结合能力相对较低, 在分析复杂的细胞和组织裂解物时, 背景的减少成为了发展过程中的一个真正的挑战。
QDB 分析可以很容易地在任何实验室采用特定抗体的访问。然而, 重要的是要指出, 抗体的特异性是一个相对的术语, 受影响的因素, 包括物种, 组织类型和细胞类型。如图 1A所示, CAPG 抗体在分析小鼠肾脏、脾脏和前列腺中的裂解液时是特异的, 但在分析小鼠肝脏时却变得不特异。事实上, 我们经常发现一种抗体是特定的一种组织类型, 但并不总是特定于其他组织类型从同一物种。因此, 先验的西方印迹分析是必要的, 以确保 QDB 分析的特殊性。事实上, 抗体的相对特异性可能是与 ELISA 分析相关的错误结果的原因, 无论制造公司花了多大力气来确保化验的特异性, 它们都不能用尽所有可能的用户可以选择使用其 ELISA 产品分析的样本类型。
与所有免疫一样, QDB 分析方法的一个潜在问题是商业抗体质量缺乏一致性。即使使用同一公司的抗体的外观质量 (相同的目录号, 等等), 也可能有大的差异, 从一个购买到另一个由于批次到批次的可变性。因此, 除非对抗体的质量有充分的信心, 否则每次购买时重新鉴定抗体是很重要的。
总之, 我们提供了一个详细的协议和一个例子, 当使用 QDB 分析方法, 以实现绝对定量测定特定的蛋白质在组织水平。结果表明, QDB 分析方法对于任何对高通量、定量应用免疫印迹分析都有兴趣的人来说是一种方便的工具。它在细胞和组织水平上实现对特定蛋白质含量的绝对测定的能力也将这种技术与传统的应用免疫印迹技术区别开来。这项功能允许对多种分析结果进行组合和比较, 这是必要的步骤, 特别是在较大的人口研究中, 以及在不久的将来在蛋白质水平上实现相关的关联研究。

随着近年来基因组研究的令人振奋的进展, 生物医学研究领域也见证了蛋白质研究的显著进展。随着生物数据在基因组和蛋白质水平上的不断积累, 利用生物信息学工具分析这些数据已成为生物医学研究的热点。因此, bioinformatical 研究的成功提高了生物和生物医学研究界对更多和更好的数据质量的需求, 只有通过基因组和蛋白水平的技术进步才能实现这一任务。
质谱 (MS) 和应用免疫印迹分析是目前蛋白质分析的两种主流技术。近年来, MS 在蛋白质组研究中占据主导地位, 能够同时对数以千计的个体蛋白质进行分析。应用免疫印迹的技术, 包括西方印迹和斑点杂交, 另一方面, 也发挥了重要作用, 在蛋白质研究, 甚至自其发明1,2,3,4, 5。酶联免疫吸附试验 (ELISA)5、6、7和反相蛋白微阵列 (RPPM)8、9可视为应用免疫印迹的高通量格式分析。然而, 所有这些免疫检测方法, 除了 ELISA, 测量的相对表达水平的特定蛋白质。这些方法的相对性质将成为人口研究的一个真正问题, 因为必须同时进行分析, 防止通过多种分析增加池大小的努力。此外, 这些研究得出的结果仅仅是半定量的, 从而使数据分析中的生物信息学努力复杂化。同时, 尽管 ELISA 非常适合于蛋白质样品的高通量绝对分析, 但由于其结合能力低, 复用10, 这种技术似乎在细胞或组织等复杂环境中遇到挑战。
我们开发了一种适合人口研究的应用免疫印迹方法, 具有方便、高通量、定量的特点, 适用于蛋白质含量的绝对测定, 并命名为该技术的定量斑点杂交分析 (QDB)11。在本研究中, 我们提出了 QDB 分析的详细协议, 并通过测定三种不同的小鼠组织, 包括肾脏, 脾脏, 前列腺的绝对蛋白质含量, 证明了该方法的 CAPG。我们认为这一详细的协议很好地说明了该方法的可行性和方便性, 为如何避免这种方法在实践中的潜在缺陷提供了指导。

<table><tbody><tr><td>Microplate reader</td><td>Tecan</td><td>Tecan Infinite 200 PRO</td><td></td></tr><tr><td>QDB plate</td><td>Yantai Zestern Co.Ltd</td><td></td><td>Plates are available upon request for verification purpose either through email or visiting www.zestern.net.</td></tr><tr><td>Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot)</td><td>jackson</td><td>&amp;nbsp;711-035-152</td><td></td></tr><tr><td>Enhanced chemiluminesence substrate</td><td>Thermo</td><td>32134</td><td></td></tr><tr><td>Rabbit anti-CAPG&amp;nbsp;</td><td>Sino Biologic Inc</td><td>14213-T52</td><td></td></tr><tr><td>CAPG protein</td><td>Sino Biologic Inc</td><td>14213-HNAE</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>Nacl</td><td>Tianjin Ruiji Jin special Chemical Co., Ltd</td><td>10461220</td><td></td></tr><tr><td>Mgcl2</td><td>Tianjin Zhiyuan Chemical Reagent Co., Ltd</td><td>20120802</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E9884-500G</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>BNN1913</td><td></td></tr><tr><td>Na2P2O7</td><td>Haituo Chinese medicine test</td><td>20160914</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G7757</td><td></td></tr><tr><td>phenylmethylsulfonyl fluoride</td><td>Sigma</td><td>P7626-5G</td><td></td></tr><tr><td>pepstatin</td><td>Sigma</td><td>Z290033</td><td></td></tr><tr><td>leupeptin</td><td>Sigma</td><td>L2884</td><td></td></tr><tr><td>aprotinin</td><td>Sigma</td><td>A3428</td><td></td></tr><tr><td>Tris-Hcl</td><td>Sigma</td><td>T6455</td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td>L5750-500G</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>43815-1G</td><td></td></tr><tr><td>Bromphenol Blue</td><td>Sigma</td><td>B-0126</td><td></td></tr><tr><td>NaF</td><td>Sigma</td><td>S7920</td><td></td></tr></tbody></table>

high throughput, absolute determination of the content of a selected protein at tissue levels using quantitative dot blot analysis (qdb)

缺乏方便、定量、高通量的应用免疫印迹方法, 在细胞和组织水平上绝对确定特定蛋白质的含量, 大大阻碍了蛋白质组研究的进展。从目前可用的应用免疫印迹技术获得的结果也是相对的, 防止任何努力将独立研究与蛋白质样品的大规模分析结合起来。在本研究中, 我们演示了定量斑点杂交分析 (QDB) 的过程, 以实现在高吞吐量格式的绝对量化。利用商业上可用的蛋白质标准, 我们能够确定在三种不同的小鼠组织 (肾脏, 脾脏和前列腺) 制备的蛋白质样品中, gelsolin (CAPG) 的上限肌动蛋白的绝对含量, 以及详细的对实验细节的解释。我们建议 QDB 分析作为一种方便、定量、高通量的应用免疫印迹方法, 在细胞和组织水平上对个体蛋白质进行绝对量化。这种方法将极大地帮助生物和生物医学研究的各个领域的标志物验证和通路验证。

确定任何蛋白质的绝对量, 包括 CAPG 蛋白在小鼠组织中需要一个特定的抗体和纯化蛋白作为标准。蛋白质标准和裂解物的分析的线性范围在任何大规模分析之前也需要建立。抗体的线性范围高度依赖于抗体本身, 并且特定抗体的适当的稀释范围需要由每个单独用户核实。
我们首先确定了小鼠肾脏、脾脏和前列腺组织中 CAPG 抗体的特异性, 使用阴性对照 (IgG 游离 BSA) 和阳性控制 (商业上可用的 CAPG 蛋白) 进行了西方印迹分析 (图 1A)。抗 CAPG 抗体是针对裂解物从小鼠脾脏, 心脏, 肌肉和前列腺 (一波段检测)。裂解物从肾脏和肝脏观察到非特异带。然而, 在肾脏裂解中, 非特异带明显弱于特异带, 表明该抗体适用于肾脏组织分析。其次, 通过两种侧向剂量曲线确定了蛋白质标准的线性范围和分析的裂解物量。根据我们过去的经验, 一种商业上可用的 CAPG 蛋白被连续稀释, 如表 1所示, 而小鼠肾脏、脾脏和前列腺的组织裂解物也根据裂解物中总蛋白含量的测定而稀释。由一个鉴定人的总蛋白测定试剂盒。两个剂量的研究并排进行, 并绘制在图 1B。根据任意单位微板块读取器测量的 QDB 信号, 选择适当量的小鼠肾、脾、前列腺裂解物。本实验的原始读数见表 3。
最后一步, 将 CAPG 蛋白标准和裂解物从小鼠肾脏、脾脏和前列腺中加载到 QDB 板上。在这种情况下, 我们选择加载0.3 µg/样品裂解物从小鼠肾脏和脾脏和1µg/样本裂解物从小鼠前列腺分析, 在这些水平上, QDB 读数是至少20倍以上的背景, 而在线性范围内的分析根据裂解物和蛋白质标准的剂量曲线。在微板块读者直接量化车牌之前, 该板块受到了描述的 QDB 协议的接受。使用串行稀释纯化 CAPG 蛋白, 我们可以建立一个剂量反应曲线, 方程, 并 R2 通过简单的回归分析使用可用的软件 (如微软办公室 excel)。用所建立的方程将小鼠肾脏、脾和前列腺裂解物的 QDB 信号转化为 CAPG 蛋白的绝对量, 结果由总蛋白量校正, 本例为裂解物的0.3 µg。老鼠脾和肾脏, 和1µg 裂解物从小鼠前列腺, 以 CAPG 蛋白的最终浓度在这些组织作为 pg/µg。结果如图 1C所示,表 4中显示了原始读数。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56885/56885fig1.jpg"> 图 1.有代表性的结果, QDB 分析, 以确定绝对 CAPG 水平的三小鼠组织 (肾, 脾和前列腺)。A.通过对脾、心、肌、肾、肝、前列腺制备的小鼠组织裂解物对兔抗 CAPG 抗体的特异性进行分析, 采用阴性对照 (IgG 游离 BSA) 和阳性控制 (商业可用 CAPG 蛋白)。用前列腺、肾、脾、纯化重组 CAPG 蛋白等裂解物制备兔抗 CAPG 抗体的线性范围 QDB 分析。结果是 triplicates 的平均值。C.从小鼠肾脏、脾和前列腺中 CAPG 含量的绝对测定裂解物。每个条形图代表单个鼠标的一个组织。结果为平均三份。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56885/56885fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 3" src="/files/ftp_upload/56885/56885table3.jpg"> 表 3: 微板块阅读器的结果从剂量研究使用连续稀释, 汇集裂解物从小鼠脾, 肾脏和前列腺和纯化的重组 CAPG 蛋白。
<img alt="Table 4" src="/files/ftp_upload/56885/56885table4.jpg">
表 4: 微板块读者的结果 QDB 分析的绝对 CAPG 水平在小鼠肾脏 (8), 脾 (4) 和前列腺 (5) 使用重组 CAPG 蛋白作为标准。

Watch this Scientific Journal Video about High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB at JoVE.com

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

在这里, 我们展示了定量斑点杂交分析 (QDB) 的详细过程, 通过确定目标蛋白的绝对含量, 帽肌动蛋白, gelsolin 样 (CAPG), 在三种不同的小鼠组织。我们展示了高通量, 方便, 定量应用免疫印迹技术的生物标志物验证在细胞和组织水平。

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

high-throughput-absolute-determination-content-selected-protein-at

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

10.9K 次观看.  Binzhou Medical University. 在这里, 我们展示了定量斑点杂交分析 (QDB) 的详细过程, 通过确定目标蛋白的绝对含量, 帽肌动蛋白, gelsolin 样 (CAPG), 在三种不同的小鼠组织。我们展示了高通量, 方便, 定量应用免疫印迹技术的生物标志物验证在细胞和组织水平。

视频: High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

科研

生物化学

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

毛细管电聚焦法对蛋白质及其亚型的高灵敏度和定量检测

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

生物学

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

V3 无污渍工作流，适用于西式印迹中实用、方便和可靠的总蛋白质加载控制

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate

Source: Qi, X., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56885">High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB).</a> J. Vis. Exp. (2018).
This video demonstrates a quantitative dot blot, or QDB, technique for the absolute quantification of proteins of interest in a tissue sample. The proteins in the sample are immobilized on the nitrocellulose membrane inside the wells of a QDB plate. Utilizing target protein-specific antibodies labeled with a peroxidase enzyme, a chemiluminescent substrate is oxidized to produce light, which is measured to determine the protein concentration of the sample.

定量斑点印迹法，用于估计组织裂解物中的靶蛋白

Source: Qi, X., et al. High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis ...

JoVE Encyclopedia of Experiments

Biological Techniques

Blot Techniques

Quantitative Western Blotting to Estimate Protein Expression Levels

For electrophoresis, set up a pre-cast 4 to 12% Bis-Tris gradient gel in the gel electrophoresis chamber system, and load 3.5 microliters of a protein standard into the well. When using an internal standard for between membrane normalization, load an amount that is equal to the other samples into the first three wells next to the protein ladder, and load 30 micrograms of each sample into the remaining wells. Then, run the samples through the stacking gel at 80 volts for 10 minutes followed by 150 volts for an additional 45 to 60 minutes.
At the end of the electrophoresis, to assemble the transfer stack, place the protein gel onto the bottom stack containing the polyvinylidene difluoride membrane followed by the filter paper. Use the blotting roller to remove any air bubbles, and place the top stack on the top of the filter paper, before rolling the stack again to remove air bubbles.
Transfer the whole stack into the transfer device with the electrode on the left side of the device and place the gel sponge on top of the stack, so that the sponge is aligned with the corresponding electrical contacts on the device.
After closing the lid, select and start the appropriate program. At the end of the program, cut the membrane to the gel size, and wash the cut membrane quickly with double-distilled water before continuing with the total protein stain.
For total protein staining, roll the membrane into a 50-milliliter tube with the protein-side facing inwards and label the membrane with 5 milliliters of protein stain solution on a roller for 5 minutes at room temperature in a fume hood.
At the end of the incubation, wash the membrane quickly with 5 milliliters of wash solution, returning the tube briefly to the roller between washes, followed by a brief rinse with ultra-pure water. Add 3 millimeters of blocking buffer to the membrane, and return the membrane to the roller for 30 minutes at room temperature. Replace the blocking buffer with the primary antibody of interest at the appropriate optimized concentration, and incubate the membrane on the roller overnight at 4 degrees Celsius.
The next day, wash the membrane 6 times for 5 minutes with 5 milliliters of fresh PBS per wash on the roller at room temperature. After the last wash, incubate the membrane with the appropriate secondary antibody solution on the roller for 1 hour at room temperature followed by 3 washes for 30 minutes per wash. After the last wash, dry the membrane, and use aluminum foil to keep the membrane protected from light.
For image acquisition, place the membrane on the scanner with the protein-side facing down and select the scanning area in the software. Then acquire images in both channels and export the images to an appropriate image analysis program.
Display the 700-nanometer channel to show the total protein staining result, and select &#34;Analysis&#34; and draw a rectangle to define the area of interest for normalization. Then, copy and paste the first rectangle area onto each individual sample to ensure the defined region is the same size for all of the analyzed lanes.
To quantify the protein concentration in each lane, copy the results from both the total protein stain and the protein of interest to a spreadsheet program, and determine the highest total protein stain signal. Then divide each total protein stain signal value by this value to obtain the normalized protein loading value, and divide the 800-nanometer signal value from each individual sample by its corresponding normalized protein value to calculate the relative protein expression ratio in different samples.