这项工作得到了研究所资助的研究补助金 DK52057 和 DK107498 从 nih 到 K.V.K. x P 得到了来自 nih 的机构培训补助金2T32DK007201 的支持。

1. 肽的处理
<ol><li>肽的设计与排序<ol>
<li>选择所需的肽序列, 并添加一个标记, 如 c-myc 表位 (EQKLISEED) 在其 N 或 C 终点。</li>
		<li>用肽溶解度计算器 http://pepcalc.com/peptide-solubility-calculator.php 检查水中肽的预测溶解度。如果溶解度低, 尝试添加另一个水溶性标签, 以改变电荷平衡。 注意: 我们使用了与第一个腔圈 (fll) 相对应的肽, 在 N 总站 (Myc-fll-GLUT4) 上标记了 GLUT4 的 c-myc 表位 (粗体): EQKLISEEDLNAPQKVIEQSYNATWLGRQGPGGPSSIPPGTLTTLWA 具有 "良好" 预测水中溶解度.</li>
		<li>订购具有至少75% 纯度的自定义肽, 这是足够的化验, 并要求提供者的溶解度测试。将该顺序分成至少两个整除数, 以防在溶解肽时出现意外问题。 注: Myc-fll-GLUT4 在两个整除数中被订购。其报告的溶解度在超纯水是≤10毫克/毫升。</li>
	</ol>
</li>
	<li>溶解肽 注: 超纯水是最好的溶剂。如果肽似乎不是水溶性的, 请参考 http://www.genscript.com/peptide_solubility_and_stablity.html 的说明。<ol>
<li>在超纯水或选择缓冲器中制备100x 的肽溶液, 浓度为100微克/毫升。</li>
		<li>将解决方案分成 50-100 µL 整除数, 并将其存储在-20 摄氏度。</li>
	</ol>
</li>
</ol>2. 细胞的处理
<ol><li>细胞培养<ol>
<li>使用野生型 (histidines) 细胞作为阴性对照, 并表达靶蛋白标记为六 (HisP) 的细胞。 注: 我们使用的 3T3 L1 前脂肪细胞稳定转染 Sortilin-c-myc/他的5。</li>
		<li>生长在 Dulbecco 的改良鹰培养基 (DMEM) 高糖的细胞, 补充10% 小牛牛血清, 谷氨酰胺 (2 毫米) 和青霉素/链霉素 (5 微克/毫升) 在37°c 在 10% CO2。</li>
		<li>坐四10厘米的细胞, 染两个与 HisP 和染其他两个与控制质粒 (野生)。48小时后转染, 细胞应达到 80-90% 融合。</li>
	</ol>
</li>
	<li>细胞裂解物的制备<ol>
<li>准备溶解缓冲 (10 毫米 Hepes, 30 毫米氯化钠, 5% 甘油, 10 毫米咪唑, 0.5% 海卫 X-100 和蛋白酶抑制剂鸡尾酒没有 EDTA 为他标记的蛋白质的隔离, pH 7.4) 并且保持它在4°c:</li>
		<li>用10毫升1x 磷酸盐缓冲盐水 (PBS) 在4摄氏度洗涤细胞三次。</li>
		<li>把盘子放在冰上, 在每道菜里加500µL 的溶解缓冲液。用细胞刮刀裂解物细胞。</li>
		<li>将细胞裂解液从每道菜放入不同的1.5 毫升管中。将管子标记为 WT-pH6、WT-pH8、HisP-pH6、HisP-pH8。</li>
		<li>将细胞裂解物通过注射器, 用26G 针向上和向下五次, 以完成裂解。</li>
		<li>离心细胞裂解物在 1.6万 x g 10 分钟在4°c。</li>
		<li>将上清液转移到新的标签相同的管。</li>
		<li>用蛋白质检测试剂盒或其他试剂盒分析蛋白质浓度。</li>
		<li>利用裂解缓冲液, 平衡所有四裂解物的蛋白质浓度。</li>
		<li>分离每个裂解液的小 (ca 20 µL) 整除, 并将其保持在-20 摄氏度, 以进行可能的控制实验和/或故障排除。</li>
	</ol>
</li>
</ol>3. 他标记的蛋白质对 HisPur 钴珠的捆绑
<ol><li>使用商用的洗涤缓冲器或准备一个 (50 毫米 Na2HPO4/NaH2PO4, 300 毫米氯化钠, 20 毫米咪唑, pH 8) 和保持它在4°c。</li>
	<li>用 tittering 洗涤缓冲液 ph 值为 8, 用 HCl 制备6的洗涤缓冲器. 保持在4摄氏度。</li>
	<li>轻轻地旋转钴珠的瓶子, 使其均匀悬浮。</li>
	<li>将40µL 的钴珠悬浮液放入四1.5 毫升管中, 标记为上述 (步骤 2.2.4)。</li>
	<li>在每管上加1毫升溶解缓冲液, 并将管子离心在 1000 x g 处, 5 秒. 吸入上清, 并添加 40 ul 的溶解缓冲液沉淀到固定的珠子上。</li>
	<li>将单元格裂解物添加到相应的管中。</li>
	<li>在 20 rpm 时, 在4摄氏度的管转子上孵育90分钟的管子。</li>
	<li>离心管在 1000 x g 5 s 和收集上清。将上清液保持在摄氏4摄氏度。</li>
	<li>添加500µL 的 ph 值8或 ph 6 洗涤缓冲器到相应的管, 并重新暂停珠轻轻。</li>
	<li>离心管在 1000 x g 5 s, 并丢弃上清液。</li>
	<li>重复步骤3.9 和 3.10, 四次。</li>
</ol>4. 肽对固定在珠子上的标记蛋白的约束
<ol><li>使用100x 工作溶液的肽 (步骤 1.2.1), 准备1x 工作解决方案, 无论是 ph 值6或 ph 8 洗涤缓冲器 (两个100µL 整除数为每个, 1 µg/毫升)。</li>
	<li>添加 100 ul 的1x 肽溶液的洗涤珠与相应的 pH 值。</li>
	<li>在 20 rpm 的管转子上孵化30分钟4摄氏度的珠子。</li>
	<li>离心管在 1000 x g 5 s, 收集上清, 并保持在-20 摄氏度。</li>
	<li>重复步骤3.9 和 3.10, 四次。 注意: 为了减少可能的背景, 以下步骤可以取代步骤 4.4 & 4.5。</li>
	<li>采取四微柱与30微米毛孔, 标签他们作为在步骤 2.2.4, 并把列在收集管。</li>
	<li>步骤4.3 完成后, 将孵化混合物转移到柱中, 允许溶液通过重力。保持在-20 摄氏度的流量。</li>
	<li>通过重力将500µL 的洗涤缓冲器与 ph 值6或 ph 8 通过相应的柱。丢弃流。</li>
	<li>重复步骤4.8 四次。</li>
	<li>在最后洗涤以后, 离心机列在 1000 x g 在十五年代。</li>
</ol>5. 钴珠洗脱
<ol><li>使用商业 Tricine 样品缓冲 (200 毫米三盐酸, 40% 甘油, 2% SDS, 0.04% 考马斯蓝色, pH 6.8) 和增加β巯基乙醇到最后浓度2%。</li>
	<li>添加40µL 的 Tricine 样品缓冲 (与β-巯基乙醇) 到洗涤珠和漩涡的样品。</li>
	<li>加热样品10分钟在100°c, 再漩涡样品, 并且离心机管子在 1000 x g 为 5 s。 注: 为了减少洗脱中可能的非特异性结合, 以下步骤可取代步骤 5.1-5.3。</li>
	<li>添加 40 ul 的洗脱咪唑缓冲器 (50 毫米 Na2HPO4/NaH2PO4, 300 毫米氯化钠, 0.25 米咪唑) 到洗涤珠, 漩涡和孵化管在室温为20分钟。</li>
	<li>离心管在 3000 x g 三十年代, 收集上清 (洗脱样品) 和转移到新的标签管。</li>
	<li>添加 40 ul 的 Tricine 样品缓冲区到每管, 加热样品10分钟在100°c, 漩涡和离心机管在 1000 x g 为 5 s。 注: 如果使用微柱, 将洗脱缓冲液添加到洗涤过的珠子上, 孵育20分钟, 并通过离心 8000 x g 进行2分钟的洗脱。 注: 样品可保持在-20 摄氏度, 直到电泳进行。</li>
</ol>6. 电泳和印迹
注: 样品已准备好, 由 SDS 页和随后的西方印迹分离。遵循凝胶电泳 (https://www-jove-com.remotexs.ntu.edu.sg/science-education/5065/the-western-blot) 协议, 并进行以下修改。
<ol><li>使用商业上可用的 10-20% Tricine 梯度凝胶。</li>
	<li>在凝胶上, 载入洗脱样品的20µL 和裂解物的20µL (步骤 2.2.10)。对于故障排除, 建议加载20µL 的未绑定材料 (步骤 3.8) 和 10 ng 的肽;在加载之前, 将相等数量的 Tricine 示例缓冲区添加到这些示例中。</li>
	<li>在商用三/Tricine/sds 运行缓冲器 (100 毫米三, 100 毫米 Tricine, 0.1% SDS, pH 8.3) 进行电泳。</li>
	<li>将材料从凝胶转移到0.45 µm 硝化膜上使用转移缓冲 (25 毫米三基, 192 毫米甘氨酸, pH 8.4) 补充20% 甲醇。</li>
	<li>室温下用3% 牛血清白蛋白 (BSA) 阻断膜, 1 小时。使用预先染色的分子量标记作为参考, 在水平上切开膜, 将顶部与 HisP 抗体杂交, 并用抗体对 c-myc 表位进行印迹, 以识别 c-myc 标记的肽。 注意: 在我们的特殊情况下, 不需要切割膜, 因为 HisP 和 Myc-fll-GLUT4 都可以用抗 c-myc 抗体检测到。</li>
</ol>7. 结果分析
<ol><li>使用图像站或 ImageJ 程序量化所有西方印迹信号的强度。</li>
	<li>在两个 pH 值的 HisP 上, 将来自 c-myc 标记肽的信号正常化为信号。</li>
</ol>

<ol>
	<li>Bogan, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glucose+transporter+translocation+in+health+and+diabetes.">Regulation of glucose transporter translocation in health and diabetes.</a> Annu Rev Biochem. 81, 507-532 (2012).</li><li>Kandror, K. V., Pilch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sugar+is+sIRVed:+sorting+Glut4+and+its+fellow+travelers.">The sugar is sIRVed: sorting Glut4 and its fellow travelers.</a> Traffic. 12 (6), 665-671 (2011).</li><li>Pan, X., Zaarur, N., Singh, M., Morin, P., Kandror, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sortilin+and+retromer+mediate+retrograde+transport+of+Glut4+in+3T3-L1+adipocytes.">Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes.</a> Mol Biol Cell. 28 (12), 1667-1675 (2017).</li><li>Kim, J., Kandror, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+luminal+loop+confers+insulin+responsiveness+to+the+glucose+transporter+4.">The first luminal loop confers insulin responsiveness to the glucose transporter 4.</a> Mol Biol Cell. 23 (5), 910-917 (2012).</li><li>Shi, J., Kandror, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sortilin+is+essential+and+sufficient+for+the+formation+of+Glut4-storage+vesicles+in+3T3-L1+adipocytes.">Sortilin is essential and sufficient for the formation of Glut4-storage vesicles in 3T3-L1 adipocytes.</a> Dev Cell. 9 (1), 99-108 (2005).</li><li>Coutinho, M. F., Prata, M. J., Alves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shortcut+to+the+lysosome:+the+mannose-6-phosphate-independent+pathway.">A shortcut to the lysosome: the mannose-6-phosphate-independent pathway.</a> Mol Genet Metab. 107 (3), 257-266 (2012).</li><li>Willnow, T. E., Petersen, C. M., Nykjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VPS10P-domain+receptors+-+regulators+of+neuronal+viability+and+function.">VPS10P-domain receptors - regulators of neuronal viability and function.</a> Nat Rev Neurosci. 9 (12), 899-909 (2008).</li><li>Mazella, 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=The+100-kDa+neurotensin+receptor+is+gp95/sortilin,+a+non-G-protein-coupled+receptor.">The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor.</a> J Biol Chem. 273 (41), 26273-26276 (1998).</li><li>Ni, X., Morales, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lysosomal+Trafficking+of+Acid+Sphingomyelinase+is+Mediated+by+Sortilin+and+Mannose+6-phosphate+Receptor.">The Lysosomal Trafficking of Acid Sphingomyelinase is Mediated by Sortilin and Mannose 6-phosphate Receptor.</a> Traffic. 7 (7), 889-902 (2006).</li><li>Westergaard, U. B., 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=Functional+organization+of+the+sortilin+Vps10p+domain.">Functional organization of the sortilin Vps10p domain.</a> J Biol Chem. 279 (48), 50221-50229 (2004).</li><li>Nykjaer, 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=Sortilin+is+essential+for+proNGF-induced+neuronal+cell+death.">Sortilin is essential for proNGF-induced neuronal cell death.</a> Nature. 427 (6977), 843-848 (2004).</li></ol>

作者没有什么可透露的。

Sortilin 是一种进化保守的多配体蛋白受体, 涉及两个信号在等离子膜和细胞内排序事件6,7。然而, 寻找正宗 sortilin 的配体 (其中一些是腔内或整体膜蛋白) 是复杂的, 因为 sortilin 与它的一些结合伙伴的相互作用似乎对洗涤剂敏感。因此, 一种简单而广泛的方法来研究蛋白质-蛋白质相互作用, 共同免疫沉淀, 不能轻易地应用。一些研究组使用免疫沉淀来演示 sortilin 及其潜在配体之间的交互作用 8, 9.然而, 这些实验是在0.1% 海卫一或0.6% 章中进行的, 这对膜增溶的完整性产生怀疑。
其他研究人员通过表面等离子体共振10,11, 研究了 sortilin 及其配体之间的相互作用。虽然表面等离子共振可以说是解决这个问题的最好方法, 但它需要昂贵的设备和大量的纯重组蛋白质, 成本高昂, 耗时。
在这里, 我们描述了一种技术, 结合其他方法, 如交叉链接和酵母两个混合系统, 强烈建议 sortilin 可以绑定到 GLUT4。该检测方法快速、简便, 可方便地适应其他蛋白质和不同大小的肽。
这项化验有几个关键步骤。第一种是肽在水中的溶解度。另一种是正确控制的选择。显然, 大量的生物材料可以与钴珠通过内生的他达到序列或非特定。因此, 在我们的案例 Sortilin 中, 必须固定在裂解物细胞和转染感兴趣蛋白质的细胞的珠子上。在这样的实验设计中, 绑定到珠子上的物质只会在一个蛋白质、Sortilin 中不同, 所以一定程度的背景结合的肽来控制珠子是可以容忍的。不过, 通过提高最终洗涤步骤的严格性, 可以减少肽对珠子的背景结合。使用迷你柱来洗涤珠子可能会进一步减少背景约束。
由于 "GLUT4 通路" 的性质, 我们希望以不同的 pH 值来解决 Myc-fll-GLUT4 对 sortilin 的约束。我们认识到, 早期/分拣内涵体的腔内 pH 值可降至6以下。然而, pH 低于6会扰乱他标记的蛋白质与钴珠子的结合, 这可能被认为是方法的限制。
用他的表位分离钴珠子上的靶蛋白, 优于其他亲和纯化步骤, 如免疫沉淀与特定的抗体, 在产量和非特定的约束力方面。然而, 完全有可能的是, 在任何其他树脂 (最好, 磁性珠) 的目标蛋白的分离, 以适当的控制将证明是成功的。

GLUT4 是一种葡萄糖转运蛋白, 主要表现在脂肪和骨骼肌细胞中, 它介导胰岛素对膳食后血糖清除的影响1。GLUT4 是一种非常稳定的蛋白质, 它是在平移后的水平上调节的。在没有胰岛素的情况下, GLUT4 在很大程度上被排除在血浆膜上 (因此葡萄糖的基底通透性较低), 主要在小胰岛素应答性囊泡 (IRVs) 和反式高尔基网络 (TGN) 的细胞内进行定位, 这可能代表IRV 捐赠舱在胰岛素的管理, IRVs 保险丝与等离子膜, 并提供 GLUT4 到其功能的地方。这增加了血浆膜的渗透率为葡萄糖, 因此葡萄糖吸收从血液入脂肪细胞和骨骼肌肌细胞膜上升10到40倍。胰岛素退出后, GLUT4 被内化成早期/排序内涵体, 然后检索到 TGN 的 IRVs 重新形成。GLUT4 路径中的两个排序步骤,即从外围早期内涵体到核周 TGN 的检索, 以及 IRVs 捐赠膜 TGN 的形成是由 Vps10p 家庭成员 sortilin 启用的, 它表示类型 i跨膜蛋白和分选受体。根据一个模型, sortilin 工作作为跨膜支架蛋白: 它结合 GLUT4 在内涵体和 TGN 的流明, 并招募 retromer 或网格蛋白适配器的细胞质侧的供体膜通过其C总站2 ,3. 这促进了 GLUT4 在胞内隔间植物常常将 GLUT4 的水泡载体的分布。
sortilin 的细胞质尾部与 retromer 和各种适配器蛋白的相互作用得到了很好的证明。然而, sortilin 对 GLUT4 (及其它的其他一些蛋白质配体) 的约束是难以证明的。特别是, 试图共同 immunoprecipitate sortilin 和 GLUT4 是没有成功的可能是由于洗涤剂敏感的性质的相互作用的这两种蛋白质。此外, 作为一种典型的转运蛋白, GLUT4 有12个跨膜域和6个腔圈, 任何组合可能代表一个 sortilin 绑定站点。同时, 大量的间接证据, 如细胞内的大量联合定位, 与膜透性 DSP 的交联, 以及酵母两种杂交系统的相互作用, 表明 sortilin 可以绑定到 GLUT4。此外, 使用后一种方法结合丙氨酸扫描诱变, 我们以前确定, Vps10p 领域的 sortilin 主要绑定到第一个腔内循环的 GLUT4。然而, 哺乳动物细胞中这种相互作用的证据却不见了。在这里, 我们已经分离了他标记的 sortilin 从转染的 3T3-L1 细胞使用钴树脂, 并证明它可以与化学合成肽相对应的第一腔内循环的 GLUT4 在 ph 值6和 ph 值 8, 类似酸性环境在TGN 膜腔内细胞膜腔和中性环境。在控制实验中, 未发现从非转染细胞中提取的萃取物在同一珠子上加载的肽结合。

<table><tbody><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8849</td><td>for use in purification of histidine tag protein</td></tr><tr><td>Phosphate-Buffered Saline&amp;nbsp;</td><td>Corning</td><td>21-040-CV</td><td>PBS</td></tr><tr><td>1mL Insulin Syringe U-100</td><td>BD</td><td>329652</td><td>26G x 1/2</td></tr><tr><td>BCA Protein Assay Kit</td><td>Pierce</td><td>23228</td><td></td></tr><tr><td>Wash buffer</td><td>Boston BioProducts</td><td>BP-234</td><td>For His-tag protein purification</td></tr><tr><td>HisPur Cobalt Resin</td><td>Thermo Scientific</td><td>89964</td><td></td></tr><tr><td>Tricine sample Buffer</td><td>Bio-Rad</td><td>161-0739</td><td>with SDS, bMercaptaethanol should be added</td></tr><tr><td>Elution&amp;nbsp; Buffer</td><td>Boston BioProducts</td><td>BP-236</td><td>For His-tag protein purificationwith 250mM Imidazole</td></tr><tr><td>Mini-Protean Tris-Tricine precast gels 10-20%&amp;nbsp;</td><td>Bio-Rad</td><td>456-3115</td><td>For seperation of peptide and small protein</td></tr><tr><td>Tris/Tricine/SDS running buffer&amp;nbsp;</td><td>Bio-Rad</td><td>161-0744</td><td></td></tr><tr><td>Transfer Buffer</td><td>Boston BioProducts</td><td>BP-190</td><td>Add 20% methanol</td></tr><tr><td>Nitrocellulose membrane&amp;nbsp; 0.45 mm</td><td>Bio-Rad</td><td>1620115</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A9647</td><td>BSA</td></tr><tr><td>Anti Myc antibody</td><td>Cell Signaling</td><td>2272</td><td>Rabbit</td></tr><tr><td>Peptide</td><td>Genscipt</td><td></td><td>customize ordering</td></tr><tr><td>Precision Plus Protein Dual color Standarts</td><td>BioRad</td><td>161-0374</td><td></td></tr></tbody></table>

detection of detergent-sensitive interactions between membrane proteins

我们探索蛋白质与蛋白质相互作用的能力是理解细胞中调控连接的关键。然而, 在许多情况下, 检测蛋白质与蛋白质之间的相互作用与重大的实验挑战有关。特别地, 排序感受器与他们的蛋白质货物在膜隔间的腔内相互作用经常以洗涤剂敏感的方式, 使这些蛋白质的共同免疫沉淀不可用。分类受体 sortilin 对葡萄糖转运体 GLUT4 的结合可以作为膜蛋白间弱腔相互作用的一个例子。在这里, 我们描述了一个快速, 简单, 廉价的检测, 以验证 sortilin 和 GLUT4 之间的相互作用。为此, 我们设计和化学合成了与 GLUT4 内腔部分的潜在 sortilin 结合表位对应的 c-myc 标记肽。Sortilin 标记六 histidines 的哺乳动物细胞, 并从细胞裂解物使用钴珠分离。在不同 pH 值下, 用多肽溶液在珠上固定 Sortilin, 用洗脱材料进行了分析。这种检测可以很容易地适应研究其他洗涤剂敏感的蛋白质-蛋白质相互作用。

裂解物是由 3T3 L1 细胞稳定转染的 Sortilin-c-myc/他的5和从野生 3T3 L1 细胞, 用作负控制.两个裂解物在 ph 值6或 ph 值8和彻底洗涤的钴珠孵化。然后在 Myc-fll-Glut4 的溶液中孵化出固定蛋白的珠子。经过仔细的洗涤, 与珠子绑定的蛋白质与0.25 米咪唑洗脱。样品被接受了, 连同原始的裂解物, 到 SDS 页在一个 10-20% Tricine 梯度凝胶跟随西部印迹与抗 c-myc 抗体, 允许检测 sortilin-c-myc 或他 (110 kd) 和 Myc-fll-GLUT4 (5 kd) (图1).
Myc-fll-GLUT4 (10 ng) 在凝胶的第一条车道被装载了作为参考。由于肽的纯度只有 75%, 污染物是明显的 (更高的波段)。原始细胞裂解物从 Sortilin 和他的表达细胞包含多波段识别的抗 c-myc 抗体。从 Sortilin 和他的表达细胞分离的材料是非常干净的。除了一些小的非特定的带, 它只有两个包含了 c-myc 的组成部分: Sortilin/他和 Myc-fll-GLUT4。在第一条车道上的污染肽似乎并没有绑定到 Sortilin/他。负控制 (洗脱液) 具有主要的非特定波段。
当 GLUT4 通过细胞内的间隔, 如内涵体和 TGN, 有不同的腔内 pH 值, 我们想评估 GLUT4 与 sortilin 在 ph 值6和 ph 值 8 (图2) 的相互作用。结果的密度测定 (未显示) 未发现 Myc-fll-GLUT4 和 Sortilin/他在不同 pH 值的珠子上保留的比例有任何显著差异。因此, 我们得出结论, GLUT4 具有与 sortilin 在内涵体和 TGN 中进行交互的能力。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57179/57179fig1v2.jpg"> 图 1.Sortilin/他与 Myc-fll-GLUT4 在钴珠上的相互作用.裂解物 3T3-L1 细胞和 3T3 L1 细胞稳定表达 Sortilin-c-myc/他 (S) 通过钴珠, 洗涤, 孵化与 Myc-fll-Glut4 在 pH 值 8, 再次洗涤, 洗脱与咪唑缓冲。Myc-fll-Glut4 (10 ng) 被装载在第一条车道作为参考。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57179/57179fig2v2.jpg"> 图 2.Sortilin/他与 Myc-fll-GLUT4 在 ph 值8和 ph 值6之间进行交互.Myc-fll-Glut4 是在 ph 值为6和 ph 8 的珠上固定在珠子上的材料, 用甘氨酸样品缓冲液冲洗和洗脱。该图已从 Pan x ( et al3) 中进行了调整。

Watch this Scientific Journal Video about Detection of Detergent-sensitive Interactions Between Membrane Proteins at JoVE.com

Detection of Detergent-sensitive Interactions Between Membrane Proteins

我们描述了一个协议, 以检测在膜蛋白之间的洗涤剂敏感的相互作用, 使用结合的排序受体, sortilin, 到第一个腔内循环的葡萄糖转运蛋白, GLUT4, 作为一个例子。

膜蛋白对洗涤剂敏感相互作用的检测

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

detection-detergent-sensitive-interactions-between-membrane

Nanyang Technological University

Research

JoVE Journal

Biochemistry

5.9K 次观看.  Boston University School of Medicine. 我们描述了一个协议, 以检测在膜蛋白之间的洗涤剂敏感的相互作用, 使用结合的排序受体, sortilin, 到第一个腔内循环的葡萄糖转运蛋白, GLUT4, 作为一个例子。

视频: Detection of Detergent-sensitive Interactions Between Membrane Proteins

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

生物工程

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

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 ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

PeptiQuick，将膜蛋白一步进生物仿入肽，用于简化蛋白质结合测定

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

生物学

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

蛋白膜覆盖含量：一个协议测试可溶性和不溶性蛋白质之间的相互作用在体外</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Fluorescence-Detection Size-Exclusion Chromatography: A Technique to Identify the Integrity of Fluorescent Membrane Proteins upon Detergent Solubilization

Source: Bird, L. E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52357">Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli</a>. J. Vis. Exp. (2015)
In this video, we demonstrate a fluorescence-detection size-exclusion chromatography technique to screen for green fluorescent protein (GFP)-fused membrane protein stability in Escherichia coli following detergent solubilization. Proteins exhibiting a symmetrical peak in the size-exclusion profile with little or no free GFP indicate purified proteins with minimum degradation and aggregation, which can subsequently be selected for structure-function analysis.

荧光检测尺寸排阻色谱法:一种在去污剂溶解后鉴定荧光膜蛋白完整性的技术

Source: Bird, L. E., et al. Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli. J. Vis. Exp. (2015)
In this ...

膜蛋白对洗涤剂敏感相互作用的检测

摘要

探索更多视频

此视频中的章节