作者感谢沃尔夫-迪特尔·斯塔尔兹博士、阿诺德·雷米博士、安东·波施博士、帕特里夏·皮亚蒂博士、汤姆·戴维斯博士、克里斯·西蒙尼博士和杰夫·德班博士对这份手稿的批判性审查和编辑。作者还感谢艾莉森·施瓦茨的技术支持。

1. 蛋白质样品准备
（描述了从细胞培养中提取蛋白质的典型程序）
<ol><li>将 HeLa 细胞培养盘放在冰中，用冰冷的三叶草盐水（TBS;20 mM 三叶草-HCl，pH 7.5，150m NaCl）清洗细胞。</li>
	<li>吸气 TBS，然后添加 1 毫升每 100 毫米菜冰冷 RIPA 缓冲 （50 mM Tris-HCl pH 8.0， 150 mM NaCl， 1% NP-40， 0.5% 脱氧酸钠， 0.1% SDS） 补充磷酸盐和蛋白酶抑制剂.</li>
	<li>使用冷塑料细胞刮刀将粘附细胞从盘子中刮下来：轻轻地将细胞悬架转移到预制的微中微管中。</li>
	<li>在旋转器上的 4 °C 下保持持续搅拌 30 分钟。</li>
	<li>在 4 °C 预制离心机中以 16，000 x g 旋转 20 分钟。</li>
	<li>轻轻地从离心机中取出管子，放在冰上。将超自然的超自然移植到冰上保存的新鲜管子上，然后丢弃颗粒。</li>
	<li>取出少量（10-20微克）的测定，以执行蛋白质检测。使用RC DC检测套件确定每个样本的蛋白质浓度。</li>
	<li>如有必要，请将蛋白质样本引用至-20 °C进行长期储存。 重复冻结和解冻周期会导致蛋白质降解，应避免。</li>
	<li>以每个样品约20微克为例，加入等量的2倍莱姆利样品缓冲区（4%SDS，10%2-麦加普托乙醇，20%甘油，0.004%溴酚蓝色，125米特里斯-HCl，pH 6.8）。</li>
	<li>在样品缓冲区中加热每个细胞裂解物 5 分钟，温度为 95 °C。</li>
	<li>离心机在16，000 x g的微中心，为期1分钟。</li>
</ol>2. 凝胶电泳与无污渍凝胶（+30分钟）
<ol><li>以标准TGX任何KD无污渍预制凝胶（中度格式凝胶），从盒式磁带底部取出梳子和胶带。</li>
	<li>将盒式磁带放在标准单元中，用 60 毫升运行缓冲器（25 mM Tris、190 m 血糖、0.1% SDS、pH 8.3）填充集成的上缓冲室。用运行缓冲器冲洗井。</li>
	<li>将下缓冲箱的每一半填充 400 毫升运行缓冲到标记的填充线。</li>
	<li>加载蛋白质样本和适当的蛋白质标记。</li>
	<li>将盖子放在油箱上，将颜色编码的香蕉塞与盖子上的相应插孔对齐。</li>
	<li>在200 V或300 V时运行凝胶约30分钟或20分钟。</li>
</ol>3. 使用化学分子 MP 系统检查蛋白质分离质量的无污渍凝胶成像（+5 分钟）
<ol><li>从细胞中取出凝胶盒。使用标准单元盖中的凝胶盒式打开工具打开盒式磁带并释放凝胶。</li>
	<li>将几毫升水涂抹在切米多克 MP 成像仪的紫外线样品盘中心。小心地将凝胶从盒式磁带上抬起，放在托盘上。</li>
	<li>启动图像实验室软件，并捕获无污渍凝胶图像 （图 1A）与以下设置： 应用：无污渍凝胶 凝胶激活时间：1 分钟 成像区域：标准凝胶 图像曝光时间：自动优化最强烈的频段</li>
	<li>从样品托盘中取出凝胶，然后立即进行转移步骤。</li>
</ol>4. 带跨布洛特涡轮系统的蛋白质转移（~10分钟）
<ol><li>打开一个跨布洛特涡轮增压米迪PVDF传输包;将底部堆栈（包括膜）放在传输盒的基座上。</li>
	<li>将凝胶放在膜上，将顶部堆叠放在凝胶上，并推出气泡。</li>
	<li>将盖子放在盒式磁带底座上，然后转动表盘将其锁定。</li>
	<li>将盒式磁带插入膨胀机舱。</li>
	<li>通过选择预设的 Turbo 程序并选择标准凝胶尺寸 （midi） 开始转移，然后按 RUN。典型的跑步只需 7 分钟。</li>
	<li>转移结束后，拆解染迹三明治，将污点和凝胶放在装有除离子水的容器中。</li>
</ol>5. 使用化学分子 MP 系统检查蛋白质转移效率和质量的无污渍凝胶和斑点成像（+5 分钟）
<ol><li>将转移后凝胶放在切米多克 MP 成像仪的样品盘上。</li>
	<li>启动图像实验室软件，并捕获传输后凝胶（图1B）的无污渍图像，设置如下： 应用：无污渍凝胶 凝胶激活时间：无 成像区域：标准凝胶 图像曝光时间：与转移前凝胶图像的曝光时间相同</li>
	<li>从样品托盘中取出凝胶，然后用以下设置对污点（图 1C）进行成像。成像时，用几滴水或TBST保持污点湿润。 应用：无污渍 成像区域：标准凝胶 图像曝光时间：自动优化最强烈的频段</li>
	<li>从样品托盘中取出印迹膜，并将其放置在具有 TBST 的容器中（TBS 中的 0.1% Tween 20）。</li>
</ol>6. 抗体孵化
<ol><li>在室温下将污点放入 TBST 中 3% 牛血清白蛋白 （BSA） 溶液中，在室温下放置 1 小时。</li>
	<li>在含有针对第一个目标蛋白和针对第二个目标蛋白质升起的兔子原抗体的溶液中，在 4 °C 的溶液中隔夜孵化污点。</li>
	<li>倒出含有主要抗体的溶液。接下来，在20毫升TBST中搅拌5分钟，洗净污点。重复4次，共洗5次。</li>
	<li>在室温下孵育1小时，在二级抗体溶液中含有Dylight 650组合山羊抗鼠抗体和Dylight 549结合山羊抗兔抗体。</li>
	<li>倒出含有主要抗体的溶液。接下来，在20毫升TBST中搅拌5分钟，洗净污点。重复4次，共洗5次。</li>
</ol>7. 图像实验室软件成像和数据分析 - 蛋白质总正常化（约 5 分钟）
<ol><li>通过打开新的多通道协议、配置三个荧光通道并运行协议，获得斑点的多重荧光图像（图 1E）。 频道 1： 应用： 污点 Dylight 650 成像区域：标准凝胶 图像曝光时间：自动优化最强烈的频段 频道 2： 应用： 污点 Dylight 549 成像区域：标准凝胶 图像曝光时间：自动优化最强烈的频段 频道 3： 应用：无污渍 成像区域：标准凝胶图像曝光时间：自动优化最强烈的波段</li>
	<li>单击分析工具框中的规范化图标，然后单击"是"以检测车道和带。</li>
	<li>如果需要，选择并使用"车道和带工具"对车道和带进行调整。</li>
	<li>选择无污渍图像作为规范化通道。</li>
	<li>选择 MW 分析工具，并通过检查下面的框来分配 MW 标准车道。</li>
	<li>要查看规范化卷，请单击工具栏上的分析表。所有计算将由软件自动执行，包括正常化因子和标准化卷。目标蛋白质带强度值现在根据蛋白质负荷的变化进行调整。这将允许在样本中准确比较目标蛋白质。</li>
</ol>

<ol>
	<li>Aldridge, G. M., Podrebarac, D. M., Greenough, W. T., Weiler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+total+protein+stains+as+loading+controls:+an+alternative+to+high-abundance+single-protein+controls+in+semi-quantitative+immunoblotting.">The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting.</a> J. Neurosci. Methods. 172 (2), 250-254 (2008).</li><li>Bauer, D. E., Haroutunian, V., McCullumsmith, R. E., Meador-Woodruff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+four+housekeeping+proteins+in+elderly+patients+with+schizophrenia.">Expression of four housekeeping proteins in elderly patients with schizophrenia.</a> J. Neural. Transm. 116 (4), 487-491 (2009).</li><li>Casta&#241;o, Z., Kypta, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Housekeeping+Proteins:+Limitations+as+References+During+Neuronal+Differentiation.">Housekeeping Proteins: Limitations as References During Neuronal Differentiation.</a> Open Neurosci. J. 2, 36-40 (2008).</li><li>Colella, A. D., Chegenii, N., Tea, M. N., Gibbins, I. L., Williams, K. A., Chataway, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Stain-Free+gels+with+traditional+immunoblot+loading+control+methodology.">Comparison of Stain-Free gels with traditional immunoblot loading control methodology.</a> Anal. Biochem. 430 (2), 108-110 (2012).</li><li>Cully, T. R., Edwards, J. N., Friedrich, O., Stephenson, D. G., Murphy, R. M., Launikonis, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+plasma+membrane+Ca-ATPase+and+stromal+interacting+molecule+1+expression+levels+for+Ca2++signaling+in+dystrophic+mdx+mouse+muscle.">Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca2+ signaling in dystrophic mdx mouse muscle.</a> Am. J. Physiol. Cell Physiol. 303 (5), (2012).</li><li>Dittmer, A., Dittmer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-actin+is+not+a+reliable+loading+control+in+Western+blot+analysis.">Beta-actin is not a reliable loading control in Western blot analysis.</a> Electrophoresis. 27 (14), 2844-2845 (2006).</li><li>Dutka, T. L., Lamboley, C. R., McKenna, M. J., Murphy, R. M., Lamb, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+carnosine+on+contractile+apparatus+Ca2++sensitivity+and+sarcoplasmic+reticulum+Ca2++release+in+human+skeletal+muscle+fibers.">Effects of carnosine on contractile apparatus Ca2+ sensitivity and sarcoplasmic reticulum Ca2+ release in human skeletal muscle fibers.</a> J. Appl. Physiol. 112 (5), 728-736 (2012).</li><li>Elliott, S., Busse, L., Swift, S., McCaffery, I., Rossi, J., Kassner, P., Begley, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+expression+and+function+of+erythropoietin+receptors+in+the+kidney.">Lack of expression and function of erythropoietin receptors in the kidney.</a> Nephrol. Dial. Transplant. 27 (7), 2733-2745 (2012).</li><li>Eslami, A., Lujan, J., Western, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blotting:+Sample+Preparation+to+Detection.">Blotting: Sample Preparation to Detection.</a> J. Vis. Exp. (44), (2010).</li><li>Ferguson, R. E., Carroll, H. P., Harris, A., Maher, E. R., Selby, P. J., Banks, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Housekeeping+proteins:+a+preliminary+study+illustrating+some+limitations+as+useful+references+in+protein+expression+studies.">Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies.</a> Proteomics. 5 (2), 566-571 (2005).</li><li>Greer, S., Honeywell, R., Geletu, M., Arulanandam, R., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Housekeeping+genes;+expression+levels+may+change+with+density+of+cultured+cells.">Housekeeping genes; expression levels may change with density of cultured cells.</a> J. Immunol. Methods. 355 (1-2), 76-79 (2010).</li><li>G&#252;rtler, A., Kunz, A., Gomolka, M., Hornhardt, S., Friedl, A. A., McDonald, K., Kohn, J. E., Posch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stain-Free+Technology+as+Normalization+Tool+in+Western+Blot+Analysis.">Stain-Free Technology as Normalization Tool in Western Blot Analysis.</a> Anal. Biochem. 433 (2), 105-111 (2013).</li><li>Hagiwara, M., Kobayashi, K., Tadokoro, T., Yamamoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+SYPRO+Ruby-+and+Flamingo-stained+polyacrylamide+gels+to+Western+blot+analysis.">Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis.</a> Anal. Biochem. 397 (2), 262-264 (2010).</li><li>Heidebrecht, F., Heidebrecht, A., Schulz, I., Behrens, S. E., Bader, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+semiquantitative+Western+blot+technique+with+increased+quantification+range.">Improved semiquantitative Western blot technique with increased quantification range.</a> J. Immunol. Methods. 345 (1-2), 40-48 (2009).</li><li>Jensen, R. B., Ozes, A., Kim, T., Estep, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kowalczykowski+SC+BRCA2+is+epistatic+to+the+RAD51+paralogs+in+response+to+DNA+damage.">Kowalczykowski SC BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage.</a> DNA Repair. , (2013).</li><li>Lanoix, D., St-Pierre, J., Lacasse, A. A., Viau, M., Lafond, J., Vaillancourt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+reference+proteins+in+human+placenta:+general+protein+stains+are+the+benchmark.">Stability of reference proteins in human placenta: general protein stains are the benchmark.</a> Placenta. 33 (3), 151-156 (2012).</li><li>Larkins, N. T., Murphy, R. M., Lamb, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influences+of+temperature,+oxidative+stress,+and+phosphorylation+on+binding+of+heat+shock+proteins+in+skeletal+muscle+fibers.">Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers.</a> Am. J. Physiol. Cell Physiol. 3 (6), (2012).</li><li>Laurie, K. J., Dave, A., Straga, T., Souzeau, E., Chataway, T., Sykes, M. J., Casey, T., Teo, T., Pater, J., Craig, J. E., Sharma, S., Burdon, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Novel+Oligomerization+Disrupting+Mutation+in+CRY&#913;A+Associated+with+Congenital+Cataract+in+a+South+Australian.">Identification of a Novel Oligomerization Disrupting Mutation in CRY&#913;A Associated with Congenital Cataract in a South Australian.</a> , (2012).</li><li>Li, X., Bai, H., Wang, X., Li, L., Cao, Y., Wei, J., Liu, Y., Liu, L., Gong, X., Wu, L., Liu, S., Liu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+validation+of+rice+reference+proteins+for+western+blotting.">Identification and validation of rice reference proteins for western blotting.</a> J. Exp. Bot. 62 (14), 4763-4772 (2011).</li><li>Liu, N. K., Xu, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-tubulin+is+a+more+suitable+internal+control+than+beta-actin+in+western+blot+analysis+of+spinal+cord+tissues+after+traumatic+injury.">Beta-tubulin is a more suitable internal control than beta-actin in western blot analysis of spinal cord tissues after traumatic injury.</a> J. Neurotrauma. 23 (12), 1794-1801 (2006).</li><li>Lowe, D. A., Degens, H., Chen, K. D., Alway, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glyceraldehyde-3-phosphate+dehydrogenase+varies+with+age+in+glycolytic+muscles+of+rats.">Glyceraldehyde-3-phosphate dehydrogenase varies with age in glycolytic muscles of rats.</a> J. Gerontol. A Biol. Sci. Med. Sci. 55 (3), 160-164 (2000).</li><li>Mollica, J. P., Dutka, T. L., Merry, T. L., Lamboley, C. R., McConell, G. K., McKenna, M. J., Murphy, R. M., Lamb, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-glutathionylation+of+troponin+I+(fast)+increases+contractile+apparatus+Ca2++sensitivity+in+fast-twitch+muscle+fibres+of+rats+and+humans.">S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans.</a> J. Physiol. 590, 1443-1463 (2012).</li><li>Murphy, R. M., Dutka, T. L., Horvath, D., Bell, J. R., Delbridge, L. M., Lamb, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ca2+-dependent Proteolysis of Junctophilin 1 and Junctophilin 2 in Skeletal and Cardiac. 591, 719-729 (2012).</li><li>Neill, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+Data+are+not+created+Equal.">All Data are not created Equal.</a> J. Clin. Invest. 119, 224 (2009).</li><li>P&#233;rez-P&#233;rez, R., L&#243;pez, J. A., Garc&#237;a-Santos, E., Camafeita, E., G&#243;mez-Serrano, M., Ortega-Delgado, F. J., Ricart, W., Fern&#225;ndez-Real, J. M., Peral, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+suitable+reference+proteins+for+expression+studies+in+human+adipose+tissue+with+relevance+to+obesity.">Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity.</a> PLoS One. 7 (1), (2012).</li><li>Rocha-Martins, M., Njaine, B., Silveira, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+Pitfalls+of+Internal+Controls:+Validation+of+Reference+Genes+for+Analysis+by+qRT-PCR+and+Western+Blot+throughout+Rat+Retinal+Development.">Avoiding Pitfalls of Internal Controls: Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development.</a> PLoS One. 7 (8), (2012).</li><li>Romero-Calvo, I., Oc&#243;n, B., Mart&#237;nez-Moya, P., Su&#225;rez, M. D., Zarzuelo, A., Mart&#237;nez-Augustin, O., de Medina, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+Ponceau+staining+as+a+loading+control+alternative+to+actin+in+Western+blots.">Reversible Ponceau staining as a loading control alternative to actin in Western blots.</a> Anal. Biochem. 401 (2), 318-320 (2010).</li><li>Said, H. M., Polat, B., Hagemann, C., Anacker, J., Flentje, M., Vordermark, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+GAPDH+regulation+in+tumor-cells+of+different+origin+under+hypoxic+conditions+in-vitro.">Absence of GAPDH regulation in tumor-cells of different origin under hypoxic conditions in-vitro.</a> BMC Res. Notes. 13 (2), 8 (2009).</li><li>Suzuki, O., Koura, M., Noguchi, Y., Uchio-Yamada, K., Matsuda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sample+mixtures+for+standard+curve+creation+in+quantitative+western+blots.">Use of sample mixtures for standard curve creation in quantitative western blots.</a> Exp. Anim. 60 (2), 193-196 (2011).</li><li>Welinder, C., Ekblad, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coomassie+staining+as+loading+control+in+Western+blot+analysis.">Coomassie staining as loading control in Western blot analysis.</a> J. Proteome Res. 10 (3), 1416-1419 (2011).</li><li>You, J., Hodge, C., Wen, L., McAvoy, J. W., Madigan, M. C., Sutton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+soybean+trypsin+inhibitor+as+an+external+loading+control+for+Western+blot+analysis+of+tear+proteins:+application+to+corneal+disease.">Using soybean trypsin inhibitor as an external loading control for Western blot analysis of tear proteins: application to corneal disease.</a> Exp. Eye Res. 99, 55-62 (2012).</li></ol>

作者安东·波施、乔纳森·科恩、肯尼思·欧、马特·哈蒙德和刘宁是生物拉德实验室公司的员工。

上述 V3 无污渍协议用于多发式荧光西印染。它也可以应用于使用化疗检测的西方印迹。在多发荧光西斑协议中，无污渍图像在两个时间点获得：1） 蛋白质转移后立即获得：2） 在抗体孵化后的多发荧光成像步骤。第一个无污渍图像用于计算传输效率，第二个无污渍图像用作加载控制。应用化疗方法时，不可能对无污渍和目标蛋白质信号进行多路复用图像，因为化疗发光信号也会出现在无污渍通道中。在这种情况下，我们建议使用蛋白质转移步骤后立即拍摄的无污渍图像进行加载控制和规范化分析。
与传统的使用家政蛋白质作为负载控制的西方污点工作流相比，V3 西部工作流程具有以下独特优势：
首先，V3 工作流程提供了实用、方便和更可靠的负载控制，以验证兴趣蛋白水平的变化。V3 协议使用总蛋白质加载控制来正常化每个样本中测量的兴趣蛋白质水平。它避免了使用家政蛋白质作为负荷控制的两个陷阱：饱和免疫检测和在某些实验条件下样品中不一致的内务蛋白质表达水平。不再需要剥离和反驳带有针对家政的抗体的步骤。使用无污渍技术，无需用 Coomassie 或 Sypro Ruby 等污渍染色和去污渍，以进行蛋白质总测量。只需几秒钟就获得污点图像，使用图像实验室软件完成蛋白质完全正常化大约需要 5 分钟。
其次，V3工作流程使科学家能够更好地控制西方程序，因为它使程序更加透明，并引入了几个质量控制检查点。在无污渍技术的帮助下，研究人员可以在凝胶和污渍上可视化他们的蛋白质样本。科学家可以评估蛋白质样本的完整性（降解与否）、分离质量（沉淀与否）、转移效率和转移质量（甚至转移与否）。这些检查点帮助研究在看到过程中存在重大缺陷时终止实验，并避免在劣质样品和斑点上浪费时间。这项技术还有助于科学家评估膜剥离后是否存在大量蛋白质损失，以及该污点是否适合反驳不同的目标4。
以下是使用 V3 西部工作流确保良好体验和优质数据的一些提示。
<ol><li>凝胶运行后立即成像凝胶。在程序中的第一次成像之前，不要将凝胶浸泡在任何缓冲器中，因为它可能会洗去污渍化合物。</li>
	<li>使用相同的成像区域获取协议中的所有图像。这将允许软件覆盖图像进行数据分析，如总蛋白质正常化。</li>
	<li>在成像前转移和转移后凝胶时，保持暴露时间一致。这将使软件能够定量测量传输效率。</li>
	<li>获取污点图像时，用几滴水或 TBST 保持膜湿润。这将避免目标蛋白检测可能出现的脏背景问题。</li>
	<li>使用低荧光PVDF膜进行多发性荧光西印。</li>
</ol>需要注意的是，紫外线激活后的无污渍分子与色氨酸残留物不可逆转地结合在一起。如果表位含有色氨酸，这种不可逆转的修饰可能会影响使用单克隆抗体时的抗原识别。多克隆抗体不太可能受到影响，因为它们识别抗原上的多个表观上位。未绑定无污渍分子很容易从凝胶和膜上冲走，因此不会干扰抗体抗原相互作用。
总之，V3西工作流程使西方污点过程更快、更透明、更定量、更可靠。研究人员现在可以很容易地在西方的污点实验中应用蛋白质总负荷控制，使他们的数据更值得信赖。V3无污渍工作流程已被一些实验室采用，其出版物已经证明，期刊接受无污渍数据作为加载控制在西方污点22，17，7，8，5，23，18，15。

西方污点是一种非常有用的技术9，然而，西方印迹有两个主要的挑战：长期和劳动密集型的过程和数据的质量。传统协议大约需要 2 天。它涉及许多步骤，包括样品制备、凝胶铸造、蛋白质电泳和转移、膜阻塞以及抗体孵化、成像，以及经常剥离、重新测试，最后进行数据分析。在整个过程中，没有可靠和灵活的流程控制工具。因此，每个步骤都可以引入错误，这些错误有可能生成数据人工制品：因此，加载控制在西方印迹中至关重要，以识别和纠正错误。加载控制通常是通过检查每个样本中参考蛋白质的蛋白质水平来完成的，以查看其是否均等。人们经常使用家政蛋白质，如β作用素 ，β-图布林，GAPDH，作为负载控制。
西方污点数据的质量取决于可靠的负载控制。但是，在使用家政蛋白进行装货控制时，有两个合理的顾虑：1） 家政蛋白带的抗体免疫检测经常饱和，因此无法区分样品之间的负荷差异：2） 在某些实验条件下，样品中的内务蛋白质表达水平可能有所不同，例如siRNA治疗、细胞死亡、细胞分化等11，28，3，6，10，21。由于这些担忧，科学期刊现在要求"对于定量比较，应使用适当的试剂、控制和具有线性信号范围的成像方法"（自然指南）。同样，《临床调查杂志》的编辑们也要求更可靠的装载控制装置24。出于这些原因，需要验证家政蛋白质才能用作负载控制。首先，必须确保它以免疫检测方法14，29的线性动态范围进行测量。第二，必须确保在所有样本26，31，25，19，20中一致表达。
可靠负载控制的另一种解决方案是使用污点中的蛋白质总量测量。一些研究人员用总蛋白污渍染色了斑点，如库马西、火烈鸟粉红、Sypro Ruby、阿米多黑、庞绍S和无污渍技术，以测量每个车道的总蛋白质信号为负载控制16，20，13，27，1，4，12。蛋白质总负荷控制避免了与家政蛋白质相关的陷阱。首先，它真实地反映了每个样本所装载的蛋白质量。其次，总蛋白污渍在用于西印分析的普通加载范围内表现出优良的线性动态范围（复合细胞赖苏酸盐的10-50微克蛋白质），并准确区分样本12的加载差异。
无污渍技术是一种新型的全蛋白染色方法，其中一种独特的化合物混合在丙烯酰胺凝胶溶液中，均匀地分布在铸造凝胶中。电泳完成后，凝胶暴露在紫外线下至少1分钟，使污渍化合物与蛋白质中的色氨酸残留物发生反应。这些蛋白质在紫外线下变得兴奋，以发出强烈的荧光信号，可以在无污渍的成像仪（如ChemiDoc MP系统）中可视化和量化。然而，无污渍化合物本身不吸收紫外线，导致凝胶图像背景低。色氨酸残留物的修饰是不可逆转的，蛋白质不仅可以在凝胶中，而且在蛋白质转移后的任何时间在污点上可视化。
无污渍技术应用于V3西部工作流程（图1），以解决对传统工作流程的主要投诉，特别是使用家政蛋白质作为负载控制的问题。使用此工作流程，可以：1） 在大约 20-30 分钟内运行凝胶：2） 在凝胶运行后 5 分钟内检查样品完整性和蛋白质分离质量：3） 在3-10分钟内转移蛋白质：4） 量化检查转移效率：和 5） 最重要的是，使用总蛋白质加载控制验证兴趣蛋白水平的变化。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>4-20% Criterion TGX Stain-Free Precast Gel</td>
 <td>Bio-Rad</td>
 <td>567-8093 </td>
 <td>Choose a gel size and percentage that meet the specific need in a experiment</td>
 </tr>
 <tr>
 <td>Trans-Blot Turbo Midi PVDF Transfer Packs</td>
 <td>Bio-Rad</td>
 <td>170-4157 </td>
 <td>Choose either PVDF or nitrocellulose and match the membrane size to the gel size</td>
 </tr>
 <tr>
 <td>Clarity Western ECL Substrate, 500 ml </td>
 <td>Bio-Rad</td>
 <td>170-5061</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>[header]</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Criterion Cell </td>
 <td>Bio-Rad</td>
 <td>165-6100</td>
 <td></td>
 </tr>
 <tr>
 <td>Trans-Blot Turbo Transfer Starter System </td>
 <td>Bio-Rad</td>
 <td>170-4155</td>
 <td></td>
 </tr>
 <tr>
 <td>ChemiDoc MP System </td>
 <td>Bio-Rad</td>
 <td>170-8280</td>
 <td></td>
 </tr>
 </tbody>
</table>

v3 stain-free workflow for a practical, convenient, and reliable total protein loading control in western blotting

西方污点是一种非常有用和广泛采用的实验室技术，但它的执行是具有挑战性的。工作流程通常被描述为"黑匣子"，因为实验者直到最后几个步骤才知道它是否已经成功执行。此外，由于在整个西方印迹处理过程中缺乏有效的质量控制工具，西方污点数据的质量有时会受到挑战。在这里，我们描述了V3西部工作流程，它应用无污渍技术来解决与传统的西方污点协议相关的主要问题。此工作流程允许研究人员：1） 在大约 20-30 分钟内运行凝胶：2） 在凝胶运行后 5 分钟内可视化样品分离质量：3） 在3-10分钟内转移蛋白质：4） 量化验证转让效率：最重要的是5）使用总蛋白质负荷控制来验证兴趣蛋白水平的变化。这种新颖的方法消除了剥离和反驳内务蛋白质的污点的需要，如β作用素、β-图布林、GAPDH 等。V3 无污渍的工作流程使西方污点过程更快、更透明、更量化、更可靠。

1. 用无污渍凝胶图像评估样品完整性、蛋白质分离质量和转移效率。
HeLa细胞中的蛋白质提取物在300 V分离20分钟，在18井标准任何KD TGX无污渍凝胶上。蛋白质样本以四种不同量装载3倍（兰斯1-3，40微克;车道 4-6， 30 μg;车道 7-9， 20 μg;车道 10-12， 10 μg）。凝胶在紫外线下激活1分钟。 图2A 显示蛋白质分离后立即获得的凝胶图像。蛋白质样本完整性（如 降解）和分离质量（如 蛋白质沉淀）可以通过此凝胶图像进行视觉评估。然后，蛋白质被转移到硝基纤维素膜使用跨布洛特涡轮增压7分钟。 图 2B 显示了转移后凝胶的无污渍图像。两张图像都是以相同的曝光时间（6.8秒）获得的。选择了3号巷和12号车道来衡量转移效率。使用图像实验室软件中的音量工具，绘制了一个矩形框（蓝色），以覆盖两个凝胶图像上的通道 3 和 12。根据这些箱子的体积值计算，两条车道的换乘效率为80%（图2C）。在这个实验中，AnyKD TGX凝胶被选中用于研究中小尺寸的目标蛋白质，并且没有针对大蛋白质的转移进行优化。优化转移效率需要使用较低的百分比凝胶（例如 4-20%）和/或调整转移时间，以促进大蛋白质的转移。2. 无污渍总蛋白负荷控制是西方印迹中家政负荷控制的可靠替代品，可以量化兴趣蛋白水平的微小变化。
MCM-7是一种DNA许可复制因子，在辐照治疗后淋巴母细胞系（LCL）中，其水平降低20-50%。在这个实验中，4个控制和辐照处理淋巴细胞系（LCL）培养物的lysates（每个30μg）在12井标准任何KD TGX无污渍凝胶上分离。凝胶在紫外线下激活1分钟，由跨布洛特涡轮转移到PVDF膜进行免疫凝血。家政蛋白GAPDH（绿色）被探测与兔子抗体（细胞信号技术，美国，1：2，500）和Dylight 549结合山羊抗兔抗体（美国罗克兰，1：20，000）。兴趣蛋白MCM-7（红色）使用小鼠抗体（美国Abcam，1：1，000）和Dylight 649结合山羊抗鼠抗体（罗克兰，1：10，000）进行探测。
图 3A 显示了在四个经过控制和辐照处理的 LCL 样本中检测到的总蛋白质（蓝色）、MCM-7（红色）和 GAPDH（绿色）的多路复用荧光图像。 图 3B 是显示每个样本中总蛋白质模式（30 μg）的相同污渍的无污渍图像。图像实验室软件选择样本通道（蓝色框）来测量 MCM-7、GAPDH 和每个通道的总蛋白质体积。MCM-7 水平在无污渍总蛋白质测量或 GAPDH 方面都进行了标准化。对规范化的MCM-7蛋白质水平进行了统计分析，并在图表（图3C）中显示MCM-7蛋白质带平均体积和标准偏差（n=4）。两种正常化方法均显示小幅下降（约25%）在MCM-7蛋白水平后进行辐照治疗。与GAPDH作为加载控制的数据相比，总蛋白质正常化的数据表现出较小的标准偏差。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50948/50948fig1highres.jpg" src="/files/ftp_upload/50948/50948fig1.jpg"> 图1。V3 西部工作流。 V3 工作流程在左列中以 4 个步骤进行描述。工作流程中使用的主要仪器和试剂在每一步都显示出来。还包括每个步骤的估计时间。右列显示在 V3 工作流中至少可生成 4 张图像。描述了每段数据的使用情况。前转移凝胶、转移后凝胶和污渍（A、B、C）的无污渍图像不能用传统的西方染色技术轻易生成：这些图像和数据提供了重要的信息和检查点沿程序，以提高科学家的控制和可重复性的西方污点工作流。如果在检测中应用了 HRP 组合的二次抗体，则目标蛋白质信号可以在化疗光斑图像（D）上捕获：如果在同一污点上同时检测多个目标蛋白，则目标蛋白信号可以捕获在荧光斑图像（E）上。
<a href="https://www.jove.com/files/ftp_upload/50948/50948fig1highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50948/50948fig2highres.jpg" src="/files/ftp_upload/50948/50948fig2.jpg"> 图2。V3 西部工作流程中转移前和转移后凝胶的无污渍图像，以评估样品完整性、分离质量和传输效率。（A） 预传递凝胶无污渍图像。（B） 转移后凝胶无污渍图像。（C） 蛋白质转移效率测量。
<a href="https://www.jove.com/files/ftp_upload/50948/50948fig2highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 3" fo:content-width="3.5in" fo:src="/files/ftp_upload/50948/50948fig3highres.jpg" src="/files/ftp_upload/50948/50948fig3.jpg"> 图3。将无污渍总蛋白质测量与 GAPDH 免疫检测进行比较，作为加载控制，以使兴趣蛋白水平正常化。（A） 荧光西斑图像。（B） 无污渍图像。（C） 标准化 MCM-7 蛋白质水平。
<a href="https://www.jove.com/files/ftp_upload/50948/50948fig3highres.jpg" target="_blank">单击此处查看更大的图像。</a>

Watch this Scientific Journal Video about V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting at JoVE.com

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

V3 工作流是使用无污渍凝胶的西方污点程序。无污渍技术使研究人员能够可视化蛋白质分离质量，验证转移效率，最重要的是，使用总蛋白质定量作为可靠的加载控制来验证兴趣蛋白的变化。

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 ...

v3-stain-free-workflow-for-practical-convenient-reliable-total

Research

JoVE Journal

Biology

22.4K 次观看.  Bio-Rad Laboratories. V3 工作流是使用无污渍凝胶的西方污点程序。无污渍技术使研究人员能够可视化蛋白质分离质量，验证转移效率，最重要的是，使用总蛋白质定量作为可靠的加载控制来验证兴趣蛋白的变化。

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

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

免疫印迹使用Invitrogen公司NuPage NOVEX双三MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

科研

生物学

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

梯级步行任务：一个评分系统，其实际应用。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

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. ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

一种实用的新的方法来提取基因组DNA保存血浆蛋白从血液采集套件

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

实用指南系统发育的非专家

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

最快在西镇：对经典蛋白质印迹分析现代风味

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...