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 border="0" cellpadding="0" cellspacing="0" style="width:911px;" width="910">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Microplate reader</td>
			<td style="width:220px;">Tecan</td>
			<td style="width:177px;">Tecan Infinite 200 PRO</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="82">
			<td height="82" style="height:82px;width:283px;">QDB plate</td>
			<td style="width:220px;">Yantai Zestern Co.Ltd</td>
			<td style="width:177px;"></td>
			<td style="width:231px;">Plates are available upon request for verification purpose either through email or visiting www.zestern.net.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:283px;">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 style="width:220px;">jackson</td>
			<td style="width:177px;">&#160;711-035-152</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:283px;">Enhanced chemiluminesence substrate</td>
			<td style="width:220px;">Thermo</td>
			<td style="width:177px;">32134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Rabbit anti-CAPG&#160;</td>
			<td style="width:220px;">Sino Biologic Inc</td>
			<td style="width:177px;">14213-T52</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">CAPG protein</td>
			<td style="width:220px;">Sino Biologic Inc</td>
			<td style="width:177px;">14213-HNAE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Hepes</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">H4034</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Nacl</td>
			<td style="width:220px;">Tianjin Ruiji Jin special Chemical Co., Ltd</td>
			<td style="width:177px;">10461220</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Mgcl2</td>
			<td style="width:220px;">Tianjin Zhiyuan Chemical Reagent Co., Ltd</td>
			<td style="width:177px;">20120802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">EDTA</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">E9884-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">EGTA</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">BNN1913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Na2P2O7</td>
			<td style="width:220px;">Haituo Chinese medicine test</td>
			<td style="width:177px;">20160914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Triton X-100</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Glycerol</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">G7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phenylmethylsulfonyl fluoride</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">P7626-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pepstatin</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">Z290033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">leupeptin</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aprotinin</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">A3428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tris-Hcl</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">T6455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">SDS</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">L5750-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">DTT</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">43815-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Bromphenol Blue</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">B-0126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">NaF</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:177px;">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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Determination of the Glycogen Content in Cyanobacteria

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

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

测定蓝细菌中的糖原含量

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

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

分光光度法测定蓝藻Synechocystis中的蛋白含量

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

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 ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

使用χCRAC以高时间分辨率监测体内蛋白质-RNA相互作用动力学

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.