Research in the Lowe-Krentz lab is supported by research grant HL54269 from the National Institutes of Health to LLK.

1. GFP蛋白质构造的设计<ol><li>购买或设计和建造，根据有关的蛋白质有GFP标记的结构。 注:对于购买构建体，标准载体可从商业实验室，包括部分或全部的以下建议:对于膜蛋白，选择的GFP的C-末端的位置，因为它不太可能与膜蛋白质运输到干扰。考虑的感兴趣的基因，如果有理由相信有关蛋白质的C-末端被紧凑地折叠成蛋白质的结构域将GFP之间的延伸。选择一般的真核细胞表达的概念，但是允许建设生产细菌系统。包括一个切割位点（如为TEV蛋白酶），通过该如果需要一些实验，其中绿色荧光蛋白可以与蛋白活性干扰GFP可以被删除。添加其它嵌如本地化或亲和力的附加标签teractions（ 例如，的His6）。这不是必需的本文所述的测定法，但可能会导致其他测定法，或者是有用的直接相邻的基因，绿色荧光蛋白之前，如果去除的GFP是期望的。 </li></ol> 2.血管细胞GFP-TMEM184A表达<ol><li>培养内皮细胞或如前面1，6，7报告了0.2％明胶包被的组织培养皿中的血管平滑肌细胞。 </li><li>加入5毫升细胞培养物胰蛋白酶溶液（0.5％重量/体积）至漂洗100mm时，或更大，细胞汇合菜。在37℃下孵育，直至细胞被刚好从板释放，并将细胞转移到无菌聚丙烯离心管中。 </li><li>添加胰蛋白酶抑制剂（ 例如，常规培养基等体积的），如在常规培养中使用，对小区/胰蛋白酶溶液，以限制胰蛋白酶活性。沉淀细胞在approxim 5分钟ately 600 XG（或适当的速度和时间，只是沉淀为标准组织培养的细胞）。吸出上清液。 </li><li>悬浮细胞在1mL HEBS（HEPES缓冲盐水）电穿孔缓冲液中，限制时间的细胞的量是在沉淀或悬浮液。放置在冰上的细胞悬浮液，并进行在冰上剩余步骤。 注:颗粒可以一次再悬浮之前被洗涤HEBS。 </li><li>添加GFP标记TMEM184A质粒足够体积，以使细胞达到20微克/毫升的DNA的终浓度。使用相同的协议GFP的构建体。 </li><li>将大约0.4毫升在试管电在HEBS细胞的解决方案。预冷却使用前的试管。 </li><li>使用以下条件电穿孔所述细胞:指数衰减，500微法，∞欧姆和170 V. 注:对于给定细胞类型的电压应被优化。与内皮细胞和平滑肌CEL初步研究升类型分别用GFP-纽构造完成，以确定这里提到的最佳电压值， 如 10。 <ol><li>为了优化电，开始与设备制造商的建议（其中包括一些标准的细胞类型条件下）。使用期望构建体或对照荧光蛋白构建体大小和荧光特性，以需要的结构相似。 注意:小型核酸似乎更容易进入细胞大于构建体，所以只用荧光蛋白的构建体可以更容易地表达比较大的一个。 </li><li>使用荧光显微镜来确定细胞生存力，其中，所述荧光构建体24和48小时后表达的细胞的百分比，和表达的强度。 注:降低电压和/或时间稍微如果生存率低。瞄准最短的时间从培养表面的细胞释放和返回之间可能细胞培养，以提高生存率。在时间上或第二脉冲略有增加可以改善结构的摄取，如果存活率高，低表达。最佳条件和表达将细胞类型之间有所不同。如果表达构建体的细胞的百分比高，但强度低，增加了DNA浓度和监测转染的细胞随着时间的推移对最佳强度，这将取决于蛋白质半衰期以及表达，可以改善强度。染色强度可以放大一些测定中，如果有必要，通过用抗GFP抗体的GFP免疫荧光染色</li></ol></li><li>电穿孔后，种子细胞成6管30毫米的组织培养孔与盖玻片成像或到组织培养皿。培养使用标准的细胞培养过程的细胞。 </li><li>可选:24小时后，更换培养基，以消除任何HEBS电缓冲区。 </li></ol> 3. GFP-TMEM可视化184A本地化<ol><li>用PBS冲洗并轻轻摇动的细胞培养至少24小时后。限制暴露于强光，避免GFP褪色。 </li><li>用4％多聚甲醛（PFA）的PBS在RT下轻轻摇动15分钟的细胞。避免使用任何含甲醇的试剂可通透细胞。如果不是必要评估配体相互作用可以使用固定用甲醇。 注意:多聚甲醛是有毒的。穿戴合适的个人防护装备。使用在通风橱中，小心，并放弃正常。 </li><li>如上用PBS冲洗。对于基于抗体染色，见下文3.6。 </li><li>摩盖玻片使用的Mowiol或其他适当的安装介质幻灯片。 </li><li>使用具有合适的过滤器集的GFP激发和发射光谱共焦或荧光显微镜图像的幻灯片。共聚焦显微镜是优选的，因为在z平面中分离的样品的能力。图像保存在曲灰度antitation目的，TIF格式（除了全色图像为插图）。 </li><li>对于通过细胞表达与WT TMEM184A比较，准备使用准备从TMEM184A序列的肽（S）一抗其他细胞免疫， 如 1。 GFP-TEMEM184A的识别，也可以使用抗GFP的完成。评估由相同的显微技术这两种细胞样本。 </li></ol> 4.罗丹明肝素结合并共定位与GFP-TMEM184A转染细胞<ol><li>治疗的GFP-TMEM184A表达细胞用100微克/毫升罗丹明 - 肝素加入到培养基中，并让细胞孵育特定的时间量，典型地为A7r5细胞少于10分钟。冲洗和修复为3.1和3.2。 注意:最佳时间和配位体的浓度依赖于细胞的反应，所得到的配位体和图像强度的荧光强度。这concentrat被采用肝素的离子，因为它导致在其他测定11的50％以上的反应。该浓度可以用标准荧光显微镜技术来可视化，因此，没有必要来增加它。罗丹明肝素与细胞中的低荧光的未标记肝素结果的稀释，因而是对图像和量化更加困难。 </li><li>定量罗丹明肝素由于GFP-TMEM184A转结合，无需转GFP-TMEM184A准备完全相同的细胞。 </li><li>图片使用共聚焦显微镜用适当的GFP（488纳米）和罗丹明（543纳米）激发和发射单元（500 - GFP的530 nm和罗丹明大于560纳米）值来确定共定位。获得至少50个细胞的图片来自至少三个独立的实验获得的统计数据。实验中保持相同的设置，并采用一个对照样品（多个）（ 例如，转染的细胞与不治疗）个在可以用来对多个实验分析标准化的数据。 </li><li>检查用的计算机程序，以确定相对于若丹明在每个细胞结合/摄取为结合定量的图像。 注意:因为它们是方便的分析，并且可以被转换为灰度在很多计算机程序，如果他们不最初保存在格式TIF图像应被采用。的Image J（免费软件）在本分析使用，并允许在图象中的任何用户定义区域被测定区域和像素强度。 <ol><li>使用手绘工具在荧光图像中圈一个单元格，使用测量工具，以确定该空间内的强度。 注意:这些测量提供所需的信息以计算总强度，用于提供一种方法，不同的小区使用不同的几何形状进行比较的用户定义的区域（全细胞，核等 ）。平均强度超过背景的区域可以报告，并有利于分析背景强度的收藏品。 </li><li>导出到一个电子表格来计算乘以强度的面积和减去背景相同数量的区域。平均足够的细胞/实验荧光/细胞获得统计学意义。 </li></ol></li></ol> 5.从GFP-TMEM184A荧光共振能量转移罗丹明肝素<ol><li>制备转染细胞并用200微克/毫升罗丹明标记的配体如在3.1孵育。注意，温育时间与荧光配体是细胞类型依赖性的。只有PFA修复，并安装幻灯片成像。 </li><li>首先，在激发405nm和图像罗丹明发射（566 - 685纳米; FRET）。其次，在激发488 GFP（493图像 - 530），第三，激发在561罗丹明（图像在566 - 685）。没有GFP-TMEM184A或不罗丹明肝素控制是至关重要的。 </li></ol> 6.罗丹明Hepari的活细胞成像吸氮量<ol><li>种子GFP-TMEM184A转染的细胞变成专为活细胞成像个别菜肴和治疗在协议3。 注意:需要的细胞的数量取决于所需的细胞类型和密度。为了最大限度地减少细胞是在悬浮液中的时间量，不计的细胞。从100毫米融合菜分为六个35毫米实时成像菜的种子细胞，以获得细胞接近汇合在48个小时。 </li><li> 48小时后，更换培养基介质无酚红。 </li><li>转移细胞的菜共聚焦显微镜用加温阶段为了保持温度，聚焦显微镜。 </li><li>吸取100微克/毫升罗丹明肝素入菜，轻轻混匀。 </li><li>立即开始录制实时图像。如果肝素没有细胞摄取鉴于区域内发生，将盘略识别细胞与含有罗丹明标记的至少一个绿囊泡。 注:成像激发和发射细节是相同的肝素摄取协议（第5）。图像之间的时间是约16秒。实验和显微镜的条件典型地将确定一个能得到图像的速度。 </li></ol> 7.从培养细胞分离GFP-TMEM184A和GFP的<ol><li>除去培养基并用PBS清洗后，加入2毫升0.2％（重量/体积）CHAPS的1×PBS溶液与蛋白酶抑制剂对表达GFP-TMEM184A或GFP（用于控制结合测定）细胞的150毫米的钢板。刮去细胞从培养皿，放置在冰上的15mL的聚丙烯管中的细胞/ CHAPS溶液，并通过敲击拌匀。完成在强光下的所有步骤，确保GFP标记漂白下面结合分析。 </li><li>特异性结合的GFP-TMEM184A（或适当GFP对照），加入2微克/毫升生物素化的抗GFP抗体的溶液中并在4℃用摇动孵育过夜。 </li><li>加入500μl的链霉亲和琼脂糖珠到至少5毫升0.2％CHAPS / PBS中并离心以大约600×g离心3至5分钟。去除上清。用新鲜的CHAPS / PBS每次重复两次以上。添加珠粒的抗体和细胞溶液中并在4℃下用摆动以允许珠粒结合到结合于GFP或GFP-TMEM184A生物素化的抗GFP抗体孵育过夜。 </li><li>通过离心（如7.3）沉淀珠子和除去未结合的材料。至少添加5mL的0.2％CHAPS / PBS /蛋白酶抑制剂洗。重复洗涤和离心至少3倍，有效地去除任何未结合的蛋白质。 </li><li>除去最后的洗涤后，（用盐酸pH至2.0）在冰上3分钟以解离抗体-GFP结合（导致自由GFP-TMEM184A或免费用1mL的0.2M甘氨酸/ 0.2％CHAPS的PBS孵育珠粒GFP）。通过点击每30秒轻轻拌匀。 </li><li>离心机在约600 XG和传送含有纯p上清rotein到一个新的15毫升管中，接着立即中和用5毫升1M的碳酸氢钠（使pH为7）。 </li><li>集中使用10,000道尔顿分子量截留离心浓缩所述样品（使用由供应商推荐离心速度）。当音量达到约0.5毫升，加0.2％CHAPS / PBS。继续浓缩，加入更多的0.2％CHAPS /直至/已添加的PBS 0.2％CHAPS的至少十体积（倍起始样品体积）的PBS。 </li><li>以确定分离的GFP或GFP标记的蛋白的量的估计值，获得的吸光度在280nm处读出，并将其与从牛血清白蛋白或其他控制蛋白质在相同缓冲液制备的标准曲线。 注:由于消光系数的差异，这种浓度的估计，但可提供近似的蛋白浓度，以方便进一步分析不浪费样本。准确的蛋白质浓度可以阻止用微型洛瑞蛋白质测定法作出某些对在相同的缓冲液作为样品制备的标准蛋白质开采。分离的蛋白质的进一步分析可以通过分离的蛋白质（使用GFP的抗体检测）和比较所预测的分子量的蛋白质印迹来实现。可替代地，使用TMEM184A抗体应导致预测的构建体大小的条带，但也可能导致一个WT TMEM184A频带如果二聚体（或更高阶低聚物）存在于样品中。纯度的估计可通过从原料的等分试样从分离的样本VS总蛋白染色总蛋白印迹来获得。 </li></ol> 8. 在体外肝素结合分析<ol><li>通过用200μL的0.2％CHAPS / PBS清洗3 5分钟倍洗涤摇动制备黑色抗生物素蛋白包被的96孔板中。准备采用相同的洗涤过程相同的黑色未涂覆的96孔板中。准备布局为实验样品（一式三份）在试验过程中要遵循。包括标准肝素浓度，缓冲控制，且与GFP样品井井无肝素确认GFP的漂白。 注意:如果优化隔离或配体相互作用缓冲剂是有问题的蛋白质不同，完成所有的板制剂和洗涤用的最佳缓冲系统。 </li><li>加入100微升60皮摩尔/孔生物素化的抗GFP的到GFP结合期望的抗生物素蛋白包被的平板的所有孔中。抗体的量是足以刚好饱和的高亲和性抗生物素蛋白孔中。 <ol><li>加缓冲器仅用于控制目的的所有孔（无GFP或GFP-TMEM184A结合需要的话）。密封井用板盖，防止水分蒸发。孵育2小时在RT，振荡。 注:加入到孔中，并温育时间的卷根据从提供者的建议。 </li></ol></li><li>清洗所有井三次5分钟用200微升0.2％CHAPS / PBS中。 </li><li>添加100微升5纳摩尔/孔的GFP-TMEM184A或GFP的拨出的抗生物素蛋白包被的板的孔中，并在室温下振荡孵育1小时。洗一遍为8.3。 注意:被选择蛋白的这一浓度，以确保所有位点用过量GFP或GFP-TMEM184A饱和。这是重要的，以确保蛋白质的相同量被绑定到每个孔中。较低量的蛋白质也可能饱和的位点，这可能通过评估孵育几个浓度的蛋白质与板和比较未结合的蛋白质和配体结合测定后残留未结合的蛋白质被检查的可能性。 </li><li>制备各种浓度的荧光素标记的肝素， 例如 ，10，25，50，75，100，200微克/毫升，在0.2％CHAPS / PBS中。不要在黑暗中这一点，所有剩余的工作。 注意:在此之前制备的浓度，为其它配体的具体浓度，应确定为protei问题N个目标。的最低浓度应在平板读数器检测的。因此，重要的是首先确定，可以准确地在所使用的缓冲系统来检测的范围内。然后确定获得可测量的结合所需要的范围内。低于10微克/毫升的荧光素 - 肝素的浓度没有一致地注册中使用的缓冲液条件下的读板器。同样，虽然如在其他测定中使用若丹明肝素，可以在板读数器可视化，在缓冲条件和用于结合所需的浓度，对标准荧光读数不与荧光团可再现不同。 </li><li>添加100μL的荧光素肝素到适当的测试孔中的抗生物素蛋白涂覆的板和浓度标准孔中都抗生物素蛋白涂覆的和未涂覆的板。孵育10分钟，在室温下振荡。板块保持在黑暗中（或覆箔）。 </li><li>使用板意图德，记录从肝素的初始荧光发射在两个板和初始荧光（总）从孔中的抗生物素蛋白涂覆的板发射与固定GFP-TMEM184A或GFP的对照孔。如果有必要，调整仪器增益，以确保最低和最高浓度之间的肝素荧光检测。 注:确保在板读取器从上方读取孔中，在孔中的最佳位置读取，如果这是可调节的。有问题的研究中，最佳位置为约75％下降的孔中。可与板读数器信息应该提供所特有的问题的平板读数器读取结合样品的测定法在黑色板的建议。 </li><li>从与固定GFP-TMEM184A或GFP的孔除去未结合的荧光肝素并放入在涂覆非黑色板相应的孔中。立即读取荧光发射。 </li><li>加100μL新鲜的0.2％CHAPS / PBS回INTØ井从中荧光肝素被删除，并读取荧光发射。 </li><li>重复步骤8.8 - 8.9的3倍。 注意:从涂覆板这些读数表明剩余在GFP或GFP-TMEM184A井荧光素肝素。如果第三未结合的样品中发现显著的荧光，应该发生的额外冲洗。如果荧光继续在每次洗涤被去除，配体的结合可能是低亲和力，和条件应重新评估。最终读数构成了束缚肝素读数。 </li><li>从取出的，未结合的，样品中的未涂覆板，获得号码每次洗涤记录荧光发射。这些都是"绑定"读数。 </li><li>使用在8.7得到的总肝素排放值，以确认肝素加入到每个孔的正确的水平。减去值平均背景读数。 注意:井无任何荧光肝素作为因抗体，GFP和缓冲的背景。 AveragE中的背景重复获得的背景值。从结合和未结合的读数减去平均背景读数得到的实际值。 </li><li>采用发射的曲线图与总荧光素肝素加入到确定发射与肝素的量的比例。 注:抗体单独，或抗体和抗原导致荧光的一些淬火。因此，总的肝素是基于如在8.7注意到总加入肝素确定。 </li><li>画出修正约束与肝素增加肝素同时适用于GFP-TMEM184A情况和GFP情况下，一式三份。 </li><li>通过加入从所有洗涤未结合的读数为特定浓度确定游离配体。 </li></ol>

<ol>
	<li>Pugh, R. J., 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=Transmembrane+Protein+184A+Is+a+Receptor+Required+for+Vascular+Smooth+Muscle+Cell+Responses+to+Heparin.">Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.</a> J Biol Chem. 291, 5326-5341 (2016).</li><li>Daher, W., 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=Identification+of+Toxoplasma+TgPH1,+a+pleckstrin+homology+domain-containing+protein+that+binds+to+the+phosphoinositide+PI(3,5)P.">Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P.</a> Mol Biochem Parasitol. , (2016).</li><li>Vit, O., 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=Large-scale+identification+of+membrane+proteins+based+on+analysis+of+trypsin-protected+transmembrane+segments.">Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.</a> J Proteomics. , (2016).</li><li>Attwood, M. 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C., 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=Heparin+responses+in+vascular+smooth+muscle+cells+involve+cGMP-dependent+protein+kinase+(PKG).">Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG).</a> J Cell Physiol. 229, 2142-2152 (2014).</li><li>Farwell, S. L., 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=Heparin+Decreases+in+Tumor+Necrosis+Factor+alpha+(TNFalpha)-induced+Endothelial+Stress+Responses+Require+Transmembrane+Protein+184A+and+Induction+of+Dual+Specificity+Phosphatase+1.">Heparin Decreases in Tumor Necrosis Factor alpha (TNFalpha)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1.</a> J Biol Chem. 291, 5342-5354 (2016).</li><li>Xu, D., Esko, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+heparan+sulfate-protein+interactions.">Demystifying heparan sulfate-protein interactions.</a> Annu Rev Biochem. 83, 129-157 (2014).</li><li>Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin/Heparan+sulfate+proteoglycans+glycomic+interactome+in+angiogenesis:+biological+implications+and+therapeutical+use.">Heparin/Heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use.</a> Molecules. 20, 6342-6388 (2015).</li><li>Slee, J. B., Lowe-Krentz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+realignment+and+cofilin+regulation+are+essential+for+barrier+integrity+during+shear+stress.">Actin realignment and cofilin regulation are essential for barrier integrity during shear stress.</a> J Cell Biochem. 114, 782-795 (2013).</li><li>Patton, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+heparin-binding+protein+using+monoclonal+antibodies+that+block+heparin+binding+to+porcine+aortic+endothelial+cells.">Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells.</a> The Biochemical journal. 311, 461-469 (1995).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP-actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP-actin.</a> J Vis Exp. , (2011).</li><li>Skalamera, D., 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=Generation+of+a+genome+scale+lentiviral+vector+library+for+EF1alpha+promoter-driven+expression+of+human+ORFs+and+identification+of+human+genes+affecting+viral+titer.">Generation of a genome scale lentiviral vector library for EF1alpha promoter-driven expression of human ORFs and identification of human genes affecting viral titer.</a> PloS one. 7, 51733 (2012).</li><li>Castro, M., Nikolaev, V. O., Palm, D., Lohse, M. J., Vilardaga, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turn-on+switch+in+parathyroid+hormone+receptor+by+a+two-step+parathyroid+hormone+binding+mechanism.">Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.</a> Proc Natl Acad Sci U S A. 102, 16084-16089 (2005).</li><li>Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F., Beltram, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+FRET+analysis+with+the+EGFP-mCherry+fluorescent+protein+pair.">Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.</a> Photochem Photobiol. 85, 287-297 (2009).</li><li>Wang, S., 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=Domain+organization+of+the+ATP-sensitive+potassium+channel+complex+examined+by+fluorescence+resonance+energy+transfer.">Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.</a> J Biol Chem. 288, 4378-4388 (2013).</li><li>Christiansen, E., Hudson, B. D., Hansen, A. H., Milligan, G., Ulven, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+a+Potent+Free+Fatty+Acid+Receptor+1+(FFA1)+Fluorescent+Tracer.">Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.</a> J Med Chem. 59, 4849-4858 (2016).</li><li>Chiang, C. F., Okou, D. T., Griffin, T. B., Verret, C. R., Green Williams, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+rendered+susceptible+to+proteolysis:+positions+for+protease-sensitive+insertions.">fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions.</a> Arch Biochem Biophys. 394, 229-235 (2001).</li></ol>

作者什么都没有透露。

此处所报告的协议被设计成提供用于鉴定TMEM184A作为在血管细胞1肝素受体确证证据。击倒技术被常规用作一种机制以确认新的蛋白质的鉴定。然而，击倒后，一些功能损失通常不是足以证明候选蛋白实际上是正确的受体（或其他功能蛋白）。同样重要的是要有证据表明该候选蛋白实际上具有的功能。用人有标签GFP的新基因的构建实现功能的实验增益和促进基于绿色荧光蛋白的亲?...

对于新功能的候选蛋白的鉴定常常取决于基于亲和力的分离方法并进行部分序列测定。新发现的蛋白质的最近的例子包括跨膜蛋白184A（TMEM184A），之后肝素亲和互动1确定了肝素受体，并TgPH1，结合磷酸肌醇PI（3,5）P 2 2 PH结构域蛋白。其他新的蛋白质鉴定涉及肽的直接序列分析，例如由维生素等。谁用跨膜肽识别蛋白产品之前未基因3。同样地，新的蛋白质序列的鉴定可以通过使用生物信息学的先前特征的蛋白质家族的搜索，如新4TM蛋白4的识别来完成的。水通道蛋白家族基因序列的考试都有人所以产生了新成员的身份与新功能5。鉴定后，蛋白质功能的分析通常是有时可以使用蛋白质功能的一个特定的测定法，诸如在水通道壳体被检查的下一个步骤。 如果可能的话，一个新发现的蛋白质的功能，可以与特定的酶或类似的体外检测功能进行检查。因为新的蛋白质的许多功能取决于只发生在完整细胞或生物体， 在体外测定法复杂的相互作用并不总是有效的。然而， 在体内测定，必须以这样的方式，它们依赖于基因序列来设计。在细胞培养和/或简单的模式生物，击倒可以提供证据为蛋白质/功能鉴定6。与如上所述确定的新的蛋白质，它通常是不足以简单地击倒的蛋白质进行确认的功能，一个d 在体内依赖于基因序列功能测定法的设计成为新的蛋白质的表征重要。 最近TMEM184A鉴定为肝素受体使用亲和层析和MALDI MS 1（其调节血管平滑肌和内皮细胞的炎性应答的增殖），7提供给开发实验的集合的机会后击倒得到的结果与识别相一致。最近的综述证实肝素与许多生长因子，它们的受体，细胞外基质组分，细胞粘附受体，和其它蛋白质8特异性相互作用。在血管系统，肝素和硫酸乙酰肝素蛋白多糖（含有在结构上与肝素类似的硫酸乙酰肝素链）与几百蛋白9相互作用。在功能上证实塔ŧTMEM184A参与了肝素吸收和结合，即用于TMEM184A基因构建技术开发的。本报告包括实验的基础上GFP-TMEM184A集合构建用于确认TMEM184A的身份肝素受体。

<table><tbody><tr><td>GFP-TMEM184A construct</td><td>OriGene</td><td>RG213192</td><td></td></tr><tr><td>Rhodamine-Heparin</td><td>Creative PEGWorks</td><td>HP-204</td><td>Light Sensitive</td></tr><tr><td>Fluorescein-Heparin</td><td>Creative PEGWorks</td><td>HP-201</td><td>Light Sensitive</td></tr><tr><td>Mowiol</td><td>EMD Millipore</td><td>475904-100GM</td><td></td></tr><tr><td>Paraformaldehyde (PFA, methanol free)</td><td>Thermo Sci Pierce Biotech, available through Fisher Scientific</td><td>PI28908 at Fisher</td><td>Use in Fume Hood</td></tr><tr><td>Reacti-bind neutravidin plates (Avidin coated black 96-well dishes)</td><td>Thermo Sci Pierce Biotech, through Fisher Scientific</td><td>PI15510 at Fisher</td><td>Pay attention to shelf-life</td></tr><tr><td>Black 96-well plates</td><td>Corning Life Sciences Plastic, purchased through Fisher Scientific</td><td>064432 at Fisher</td><td></td></tr><tr><td>A7r5 vascular smooth muscle cell line</td><td>ATCC</td><td>CRL 1444</td><td>Can be exchanged into MEM medium&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>BAOEC bovine aortic endothelial cells</td><td>Cell Applications, Inc.</td><td>B304-05</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;1,7&lt;/sup&gt;</td></tr><tr><td>BAOSMC bovine aortic smooth muscle cells</td><td>Cell Applications, Inc.</td><td>B354-05</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>RAOEC rat aortic endothelial cells</td><td>Cell Applications, Inc.</td><td>R304-05a</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;7&lt;/sup&gt;</td></tr><tr><td>Biotinylated anti-GFP</td><td>Thermo Sci Pierce Biotech, through Fisher Scientific</td><td>MA5-15256-BTIN</td><td></td></tr><tr><td>Streptavidin-coated beads</td><td>Sigma</td><td>S1638</td><td></td></tr><tr><td>HeBS</td><td>Available from Bio-Rad</td><td></td><td>Can be prepared in the lab. The pH is 6.8</td></tr><tr><td>TMEM184A antibody to the N-terminus</td><td>Santa Cruz Biotechnology</td><td>sc292006</td><td>Only known TMEM184A antibody to N-terminal region.</td></tr><tr><td>TMEM184A antibody to the C-terminus</td><td>Obtained from ProSci Inc, Poway, CA&amp;nbsp;</td><td>Pro Sci 5681</td><td>ProSci used in &lt;strong&gt;Figure 1&lt;/strong&gt;</td></tr><tr><td>GFP antibodies</td><td>Santa Cruz Biotechnology</td><td>sc9996</td><td>Used in &lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Secondary antibodies, labeled with TRITC or Cy3</td><td>Jackson ImmunoResearch Laboratories, Inc, West Grove, PA</td><td>711 025 152 (donkey anti-rabbit, TRITC)&lt;br/&gt; 715 165 150 (donkey anti-mouse, Cy3)</td><td>Minimal cross-reactivity to minimize any nonspecific staining.</td></tr><tr><td>CHAPS</td><td>Purchased from Sigma</td><td>C5849</td><td>Note that this specific catalog number has been discontinued.&amp;nbsp; Supplier will provide information regarding replacement.</td></tr><tr><td>Live imaging 35-mm dishes</td><td>MatTek (Ashland MA)</td><td>P35G-1.0 &amp;ndash; 20 mm - C</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 510 Meta with a 63X oil-immersion lens</td><td>Used for images and live-imaging in &lt;strong&gt;Figures 1, 2 and 3&lt;/strong&gt;</td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>C2+ confocal with a 60X oil-immersion lens</td><td>Used for images in &lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>Zeiss LSM 880 with a 63X oil-immersion lens</td><td>Used for images in &lt;strong&gt;Figure 2C&lt;/strong&gt;</td></tr><tr><td>Electroporation equipment</td><td>Bio-Rad</td><td>Gene Pulser X-Cell System</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Available from MidSci</td><td>EC2L</td><td>Can also be obtained from equipment supplier</td></tr><tr><td>Plate reader</td><td>TECAN</td><td>TECAN Infinite&amp;reg; m200 Pro plate reader</td><td>Readings in the middle of the wells rather than at the surface.</td></tr><tr><td>Computer program for measuring staining intensity</td><td>Image J</td><td>https://imagej.nih.gov/ij/&lt;br/&gt; Program and information available on-line</td><td>Any appropriate program can be used. See https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html for additional detail&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Cell Culture trypsin solution</td><td>Sigma</td><td>T4174</td><td>purchased as a 10x solution</td></tr></tbody></table>

using a gfp-tagged tmem184a construct for confirmation of heparin receptor identity

当新的蛋白质通过基于亲和力的分离和生物信息学分析发现，他们往往在很大程度上未鉴定。针对预测序列中的特定肽抗体允许一些本地化实验。然而，与抗体的其他可能的相互作用往往不能排除。这种情况提供给开发一套依赖于蛋白质序列测定的机会。具体地，得到含有耦合到在蛋白质的C末端的绿色荧光蛋白编码序列的基因序列的构建体和用于这些目的。实验以表征本地化，配体亲和力和功能的获得最初设计和执行，以确认TMEM184A的鉴定为肝素受体1。此外，该构建体可用于研究，处理膜拓扑问题，并详细蛋白质 - 配体相互作用。本报告介绍AR的基础上在血管细胞中表达的GFP-TMEM184A构造，可以很容易地适用于其他的新的蛋白质实验方案法兰。

而，在理论上，任何DNA构建体转染到细胞中可以与亲脂转染试剂来完成，以前的报道显示GFP的更有效的转染构建成利用电12内皮细胞。这里提供的协议典型地实现了使用的主要来源的内皮细胞和平滑肌细胞的大于80％的GFP构建表达。使用时可能会迅速提供这种构造的商用系统所采用的结构设计。主要用途集中于位置的问题，因此，正确传递到膜，具有最佳...

Watch this Scientific Journal Video about Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity at JoVE.com

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

与羧基末端设计用于真核表达GFP的标签的构建编码TMEM184A，在旨在证实TMEM184A的鉴定如在血管细胞肝素受体测定中使用。

用GFP标记TMEM184A构建肝素受体身份确认

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

using-gfp-tagged-tmem184a-construct-for-confirmation-heparin-receptor

Research

JoVE Journal

Genetics

7.9K 次观看.  Lehigh University. 与羧基末端设计用于真核表达GFP的标签的构建编码TMEM184A，在旨在证实TMEM184A的鉴定如在血管细胞肝素受体测定中使用。 

视频: Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

科研

生物化学

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

生物工程

<meta charset="utf8"></head><body>使用 CRISPR/Cas9 敲入在斑马鱼幼虫中进行荧光蛋白标记

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>荧光标记以可视化斑马鱼中的低表达蛋白质

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

荧光标记以可视化斑马鱼中的低表达蛋白质

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

遗传学

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

神经科学

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

生物学

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

用GFP标记TMEM184A构建肝素受体身份确认

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测量TCR-的pMHC结合

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

成像蛋白质 - 蛋白质相互作用在体内

基因编码的分子探针来研究G蛋白偶联受体

重组离子通道的相对细胞表面表达的总用流式细胞仪测定