我们感谢迈克尔 Heymann 和弗兰克 Siedler 生产硅片, 核心设施 MPI-B 协助蛋白质纯化和西蒙克雷奇默和莱昂哈灵顿评论的手稿。

1. 蛋白质生产
<ol><li>蛋白表达<ol>
<li>转化大肠杆菌BL21 (DE3) pLysS 以各自质粒为表达头脑6, EGFP 头脑29, mRuby3-MinD24, 矿6或 MinC30。对于质粒图, 请参阅补充资料。</li>
		<li>使用各自的抗生素 (例如,100 µg/毫升氨苄西林或50µg/毫升卡那霉素) 在 LB 培养基上接种一夜文化, 并在37摄氏度时孵育14-16 小时, 同时晃动。</li>
		<li>接种500毫升结核培养基, 含有各自的抗生素与隔夜文化 (1:200 稀释) 和孵化培养37摄氏度, 而颤抖在 180 rpm。</li>
		<li>当文化达到 600 nm 0.5-0.7 的光密度时, 通过添加0.5 毫米 IPTG 诱导蛋白表达。在 EGFP 或 mRuby3-MinD 的情况下, 将细胞转变为16°c 的孵化器, 生长细胞为14-16 小时, 在 MinC、心或矿的情况下, 在诱导后将细胞培养为3-4 小时37摄氏度。 注: 诱导 MinC, 心或矿表达对细胞有毒, 因为过度表达导致细胞分裂缺陷;因此, 重要的是37摄氏度的潜伏期保持在4小时以下。如果需要更多的蛋白质, 增加的数量的文化, 但不是孵化时间。</li>
		<li>在各自潜伏期以后收获细胞通过离心在 4000 x g 为10分钟并且存放细胞药丸在-80 °c 直到进一步用途。</li>
	</ol>
</li>
	<li>蛋白质纯化 注: 蛋白质可以在自动蛋白质纯化系统上使用预镍 NTA 柱纯化, 也可使用镍 NTA 珠进行重力流工作台提纯。<ol>
<li>为纯化与预镍 NTA 柱在自动蛋白质纯化系统使用缓冲 A1 (50 毫米磷酸钠 pH 值 8.0, 500 毫米氯化钠, 10 毫米咪唑), 缓冲 B1 (50 毫米磷酸钠 pH 值 8.0, 500 毫米氯化钠, 20 毫米咪唑) 和缓冲 C1 (50 毫米钠磷酸盐 pH 值 8.0, 500 毫米氯化钠, 250 毫米咪唑。用镍 NTA 珠进行重力流工作台的净化使用缓冲 A2 (50 毫米三盐酸 pH 8.0, 300 毫米氯化钠, 10 毫米咪唑), 缓冲 B2 (50 毫米三盐酸 pH 值 8.0, 300 毫米氯化钠, 20 毫米咪唑), 和缓冲 C2 (50 毫米三盐酸 ph 值 8.0, 300 毫米氯化钠, 250 毫米咪唑)。用10毫米ß巯基乙醇或0.4 毫米 TCEP (三 (2-羧乙基) 膦) 作为还原剂, 补充所有缓冲器。</li>
		<li>并用重悬细胞在20-30 毫升的缓冲 A1 或 A2 补充 EDTA 游离蛋白酶抑制剂, 100 µg/毫升溶菌酶, ~ 250 U/毫升 DNase 和0.2 毫米毫克2 +-adp (mg2 +-adp 在头脑或 EGFP 头脑净化仅, 从100毫米 adp 股票在100毫米氯化镁2与 pH 值调整了到 7.5)。</li>
		<li>溶解细胞使用尖端 sonicator (30% 振幅, 2.5 分钟, 三十年代脉冲, 三十年代关闭), 同时保持在冰浴中含有细胞的瓶子。</li>
		<li>通过离心细胞裂解物, 在 2.5万 x g 和4摄氏度的45分钟内去除细胞碎片。</li>
		<li>在镍 NTA 柱或镍 NTA 珠上孵化上清。<ol>
<li>对于预镍 NTA 柱, 使用自动蛋白质纯化系统的采样泵将样品装载到柱上。</li>
			<li>对于台式的纯化, 在4摄氏度的旋转振动筛上, 用镍 NTA 珠在50毫升的反应管内孵化样品1小时。对于后续步骤, 使用25毫升吸管将镍 NTA 珠转移到空柱上。</li>
		</ol>
</li>
		<li>使用至少5列缓冲 A1 或 A2 的卷进行清洗。</li>
		<li>使用至少5列缓冲 B1 或 B2 的卷进行清洗。</li>
		<li>洗脱蛋白与缓冲 C1 或 C2。</li>
		<li>通过 SDS 页评估蛋白质纯度。</li>
		<li>可选: 进一步纯化蛋白质, 将其应用于凝胶过滤柱平衡在贮存缓冲器中 (50 毫米 HEPES/KOH pH 7.2, 150 毫米氯化钾, 10% 甘油, 0.1 毫米 EDTA, 0.4 毫米 TCEP, (0.2 毫米毫克2 +-ADP 在考虑的情况下))。 注: 推荐采用凝胶过滤, 以去除蛋白质的聚集部分。</li>
		<li>如果不使用凝胶过滤, 用重力流脱盐柱 (见材料表), 将镍 NTA 洗脱缓冲液 (50 毫米 HEPES/KOH pH 7.2, 150 毫米氯化钾, 10% 甘油, 0.1 毫米 EDTA, 0.4 毫米 TCEP, (在考虑情况下0.2 毫米镁-ADP))。</li>
		<li>在液态氮中整除数的休克冷冻蛋白, 贮存在-80 摄氏度, 直到进一步使用。</li>
		<li>用布拉德福德测定法测定蛋白质的浓度, 用 atp 酶法测定蛋白质活性20。 注意: 不要用 280 nm 的吸收来评估蛋白质浓度。在心灵净化过程中核苷酸的存在和我的令人心旷神怡的缺乏扭曲了280浓度测量。使用布拉德福德或评估方法来测量蛋白质浓度。</li>
	</ol>
</li>
	<li>蛋白质标签 注: 荧光蛋白与小蛋白矿的融合, 对其扩散特性和功能有较大的改变;因此, 化学标记的蛋白质 (半胱氨酸在位置 51) 是首选超过融合到荧光蛋白。<ol>
<li>在5-10 µL 亚砜 (二甲基亚砜) 中溶解0.125 毫克的 maleimide 染料共轭, 并在0.5 毫升矿整除中加入, 在 pH 为7.2 的贮存缓冲中, 如上文详述。</li>
		<li>在温和摇动或搅拌下, 在4摄氏度或2小时内孵育2小时至一夜。</li>
		<li>分离染料和蛋白质使用重力流动脱盐塔平衡与存贮缓冲 (50 毫米 HEPES 或 KOH pH 值 7.2, 150 毫米氯化钾, 10% 甘油, 0.1 毫米 EDTA, 0.4 毫米 TCEP)。</li>
		<li>为了进一步去除任何不独立的染料, 透析蛋白质, 以防止过量的存储缓冲。</li>
		<li>通过测量各自染料的最大灭绝量并计算估计的标签效率, 验证成功的标签。有关评估标签程度的详细协议, 请参阅染料制造商的说明。用 SDS 页进行分析, 用质谱法确定总质量, 以获得样品均匀性和标记成功的进一步有用信息。</li>
	</ol>
</li>
</ol>2. 小 Unilamellar 泡 (SUV) 准备
<ol><li>multilamellar 泡的产生<ol>
<li>计算所需混合物和最终 SUV 容积中氯仿的脂量。浓度应为4毫克/毫升的脂在最小缓冲 (25 毫米三盐酸 pH 值 7.5, 150 毫米氯化钾, 5 毫米氯化镁2)。为标准 Min 试验 7:3 DOPC 的混合物: DOPG (摩尔百分比) 推荐。当使用大肠杆菌极性脂质提取物时, 使用 SLB 缓冲 (25 毫米三盐酸 pH 7.5, 150 毫米氯化钾) 进行所有准备步骤。 注意: 建议用户首次使用大肠杆菌极性脂质提取物, 因为这种混合物产生的同源 SLBs 更具挑战性。</li>
		<li>使用一个积极的位移吸管与玻璃小贴士, 混合在氯仿的脂质在一个1.5 毫升的玻璃瓶。<ol>
<li>在缓慢转动瓶子的同时, 在轻微的氮气流下干燥油脂。将脂质置于更强的氮气流下10到20分钟。将含有干脂膜的瓶子放在真空干燥中, 并将真空度至少1小时。</li>
		</ol>
</li>
		<li>水化涡流室温下的脂质, 直到混合物均匀不透明。 注: 对于 multilamellar 囊泡产生的小 unilamellar 囊, 脂质可以挤压如2.2 所述。或微气泡如2.3 所述。一般来说, 挤压产生一个较小的大小分布, 有助于形成支持的脂双层。</li>
	</ol>
</li>
	<li>用挤出机制备 SUV<ol>
<li>通过冷冻解冻7到10个循环, 打破脂质团聚体和 multilamellar 结构, 进一步溶解脂质。<ol>
<li>用 70° c 到 99° c 上的热板和带有液氮的容器准备一个烧杯。</li>
			<li>用大镊子将瓶子放在液氮中, 直到氮气停止沸腾。然后把瓶子转移到热水中, 直到溶液完全解冻。重复这些步骤, 直到脂质混合物看起来清晰的眼睛, 取决于混合物。</li>
		</ol>
</li>
		<li>组装脂质挤出机, 用最小缓冲器预冲洗系统。通过50纳米孔径的膜将脂质混合物挤出35到41倍。请确保以奇数的刀路结束, 以避免从未穿过膜的聚合。</li>
	</ol>
</li>
	<li>超声波 SUV 准备<ol>
<li>为了更好地溶解脂质在缓冲, 把包含溶液的玻璃瓶在一个热量块设置为37°c 和漩涡每20分钟1分钟。总孵化约1小时。</li>
		<li>在 sonicator 浴 (在这项工作1.91 升, 80 W) 浸泡瓶子的底部, 通过附加到一个夹具立场, 在所需的高度。</li>
		<li>在 sonicator 浴中设置水的高度, 使小瓶周围的溶液被脉冲彻底搅动, 并油脂实验脂质混合物大约20分钟。通过评估血脂的清晰度来检查成功的超声波。</li>
	</ol>
</li>
	<li>suv 可以储存在4°c 长达一周或冻结在-20 °c 在小整除数 (~ 20 µL) 和存储几个星期。解冻瓶或管在室温和油脂实验再次按照2.2.3 或5.3 的描述, 直到解决方案是明确的之前, 使用 suv, 以准备支持的脂双层 (SLBs)。请注意, 挤压后所获得的 suv 的窄尺寸分布在冷冻后和随后的解冻和超声波后丢失。</li>
</ol>3. 清洗玻璃盖玻片
注意: 玻璃盖玻片的清洗和亲水化是均匀和流体支持的脂质双层的一个重要因素。玻璃盖玻片可以使用食人鱼溶液清洁, 由7:2 硫酸的比例, 50% 过氧化氢 (3.1), 或与等离子清洁剂 (3.2) 的氧等离子体。两种方法产生相似的结果。
<ol><li>食人鱼清洁盖玻片<ol>
<li>应用食人鱼解决方案<ol>
<li>在倒置的玻璃培养皿或其他惰性表面上分发玻璃盖玻片。用玻璃吸管, 加入7滴浓硫酸 (98%) 到每个盖玻片的中心。 注意: 硫酸具有强烈的酸性和腐蚀性。工作在通风柜和适当的保护设备只。</li>
			<li>在酸滴中间添加两滴50% 的过氧化氢。 注意: 过氧化氢对眼睛和皮肤有腐蚀性。</li>
			<li>覆盖反应和孵化至少45分钟。 注意: 这里的最大等待时间对实验结果并不重要, 可以延长到数天。</li>
		</ol>
</li>
		<li>清洗食人鱼清洗盖玻片。<ol>
<li>用镊子单独拿起盖玻片, 用超纯水冲洗掉酸。将洗涤过的盖玻片放在不粘持者或类似的运输装置中。</li>
			<li>用超纯水冲洗每个盖玻片, 用加压气体 (氮气, 空气, 只有无油) 干燥表面。用永久性标记标记盖玻片的清洗面。</li>
		</ol>
</li>
	</ol>
</li>
	<li>等离子清洗盖玻片<ol>
<li>用过量的乙醇冲洗盖玻片, 之后再用超纯水。用加压气体干盖玻片。装配室如4所述。</li>
		<li>在室大会以后描述在4采取盖玻片与附有的房间和地方在血浆清洁器与氧气作为过程气体。清洁盖玻片与血浆 (在这工作30% 力量, 0.3 毫巴氧气压力为1分钟使用了)。在 SLB 形成前进行清洗, 如5所述, 随着时间的推移, 等离子清洗的 hydrophilizing 效应逐渐消失。 注: 等离子清洗的定时和功率应使用荧光标签膜进行优化, 因为太少或过量的等离子清洗既会导致不动膜或有孔的膜。</li>
	</ol>
</li>
</ol>4. 会议厅大会
<ol><li>用锋利的剪刀, 切断并丢弃0.5 毫升反应管的盖子和锥形部分。将 UV 胶水涂抹在管子的上边缘, 用吸管尖端均匀分布。</li>
	<li>把管子倒过来涂到以前清洗过的盖玻片上。在食人鱼清洗时, 一定要把它粘在玻璃的清洁面上。通过在 360 nm 的灯下放置多个腔或 LED 5 到15分钟来固化 UV 胶。</li>
</ol>5. 支持的脂质双层 (SLB) 形成
<ol><li>预热块到37°c 和孵化2毫升反应管与最小或 SLB 缓冲器, 1 管每室。</li>
	<li>将氮气吹入组装和固化的腔内, 除去在 UV 固化和组装过程中可能已沉淀的灰尘或其他颗粒。等离子清洗如3.2 所述, 如果你没有清洁你的盖玻片与食人鱼解决方案 (3.1)。把房间放在热块上。</li>
	<li>稀释20µL 整除的透明脂 (在4毫克/毫升) 与130µL 的最小缓冲或 SLB 缓冲的情况下,大肠杆菌极性脂质提取物, 产生的工作浓度为0.53 毫克/毫升。如果脂质被冷冻, 油脂实验先把管子放进浴缸 sonicator 再加入缓冲液, 然后再油脂实验缓冲。</li>
	<li>将75µL 的脂质混合物添加到每个腔室, 并将计时器设置为3分钟 (用于 DOPC/DOPG 混合物; 对于其他脂质混合物, 更长的孵化可能是必要的)。如果大肠杆菌极性脂质提取物, 吸管 CaCl2从一个100毫米的股票到房间的最后浓度3毫米。在孵化时间内, 囊泡在亲水性玻璃表面爆裂, 熔丝形成一个相干 SLB。</li>
	<li>60秒后, 在每个会议厅中添加150µL 的最小缓冲区。</li>
	<li>洗涤室: 再过120秒 (3 分钟), 通过添加200µL 或 SLB 缓冲器, 仔细吹打上下几次, 取出并添加另200µL, 以洗涤每个房间。<ol>
<li>每室洗一次后, 进行彻底清洗, 直到2毫升的缓冲用完。洗涤 SLBs 需要一些经验, 以完善的范围内的议案在会议厅, 并找到正确的洗涤强度。 注: 切勿从室中取出所有液体, 以免 SLB 干燥。 注: 在洗涤的顶端, 膜的性质将取决于许多其他因素: 脂类的类型和他们的相对浓度脂质混合物, 为 suv 的准备方法, 表面处理和事先清洁的支持。</li>
	</ol>
</li>
</ol>6. 自组织分析法
<ol><li>将燃烧室内的缓冲容积调整为200µL 最小缓冲量减去蛋白质和 ATP 溶液的数量, 然后添加头脑, 标记头脑, 我的, 如果需要, MinC。通过吹打轻轻混合元件。例子集中是1µM 头脑 (掺杂与 30% EGFP 头脑), 1 µM 矿 (掺杂与10% 个化学上标记的矿) 和0.05 µM MinC, 但样式形成在范围的集中6,10,20,30.</li>
	<li>添加2.5 毫米 atp (从100毫米 atp 股票100毫米氯化镁2, pH 7.5) 开始自组织的 MinDE。 注: 组分加法的顺序 (头脑、矿和 ATP) 可以是不同的, 并且不会影响最后的样式结果4。</li>
	<li>观察 MinDE 在荧光显微镜上的自组织 (见材料表)。MinDE 自组织也可以观察使用 TIRF 显微镜。对于成像 eGFP, 使用 488 nm 氩激光或可比二极管激光器 (例如, 490 nm)。对于成像 mRuby3-MinD, 最好使用561纳米二极管激光器。 注意: 避免高水平的励磁, 因为我们和其他17观察到的毒性在 MinDE 系统, 导致不可逆的蛋白质聚合在膜上。</li>
</ol>7. 微结构
注: 烷是一种聚合物, 可用于生产显微组织和微流控装置。一个图案化的硅晶片作为一个模子为铸造的微结构。然后, 该结构为 SLB 的形成和检测设置提供了支持。
<ol><li>要么生产硅晶片与 microcompartments 自己使用光刻 (见 Zieske 和 Schwille 详细的协议31或 Gruenberger等等视频协议32) 或订购您所需的硅片从铸造.有关本片使用的晶片图案, 请参阅补充资料。</li>
	<li>从有图案的硅片中生产出的微结构。<ol>
<li>用塑料杯重10克的基部和1克的聚硅烷交联剂。要么使用混合设备混合和加气的混合, 或手动混合, 然后德加在真空下。</li>
		<li>使用吸管尖端直接将少量的有机硅烷放到硅片上的结构上。 注意: 小心不要刮掉硅片。</li>
		<li>立即放置一个 #1 盖玻片上的一滴, 并采取干净的吸管尖端的上端轻轻地按盖玻片到硅片上。应在盖玻片和硅片之间分散。</li>
		<li>将晶片与盖玻片放入烤箱, 并在75摄氏度的3-4 小时或一夜之间治疗。从烤箱中取出晶片, 让它冷却到室温。用剃刀刀片, 小心地删除盖玻片与所附的硅晶片。 注意: 为了防止硅片变得脏或损坏, 总要覆盖微结构和盖玻片。然而, 由于在微结构中出现裂纹, 因此不使用超过三周的非晶硅结构。</li>
	</ol>
</li>
</ol>8. 微结构中的自组织
<ol><li>使用盖玻片的显微组织连接一个房间, 如4所述。</li>
	<li>清洁和 hydrophilize 表面在氧气等离子清洗器, 如3.2.2 下所述。不要食人鱼清洁的基材。</li>
	<li>建立一个 MinDE 的自组织分析, 如下所述6。</li>
	<li>在实验建立后, 检查常规的 MinDE 模式形成, 并在荧光显微镜上正确形成显微组织。</li>
	<li>当常规的 MinDE 模式形成 (10-30 分钟), 轻轻地上下两次, 以混合组件, 然后删除缓冲一步一步的吹打。使用100µL 吸管取出大量的缓冲液, 然后用10µL 吸管小心地取出其余的缓冲器。 注意: 这一步可能需要一些练习。如果太多的缓冲被取出或过程花费太长, 组织将被烘干;如果服用太少, 蛋白质不会被局限在微结构中, 而是继续形成行进的表面波。</li>
	<li>立即关闭室与盖子, 以避免干燥的残余缓冲在微结构。</li>
	<li>为了让成像时间更长, 在房间内插入一块湿润的海绵, 然后用盖子关闭。确保海绵不接触盖玻片表面。</li>
	<li>在对表面进行显微组织检查之前, 缓冲液已经足够的降低, 使表面上的微结构 MinDE 模式形成停止。显微组织中的图像 MinDE 振荡。检查显微组织是否在成像过程中不干燥或正在枯竭。 注: 每次使用后丢弃盖玻片, 如 microcompartments 的裂纹。</li>
</ol>9. MinDE 模式形成分析
<ol><li>在平面支持的脂质双层上量化 MinDE 自组织的波长、波速和波形剖面。斐济与标准成套的插件是充足的为基本的分析33。</li>
	<li>通过获得 kymographs 和时间平均蛋白质浓度剖面分析 microcompartments 中的极-极振荡。在斐济, 可以通过重新切片沿线选择的时间序列来获得基本 kymographs。</li>
</ol>

<ol>
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作者没有什么可透露的。

本文以棒状的 MinCDE 组织为例, 描述了平面支撑脂质双层和脂双层覆盖3D 结构体外重建的一种协议。为了从这些化验中获得有价值的数据, 最重要的控制因素是蛋白质和膜质量。
为了保证蛋白质质量, 应使用 SDS 页和质谱法确认蛋白质的含量。此外, 通过分析凝胶过滤或动态光散射, 验证蛋白质是可溶的, 而不是聚合的。凝胶过滤可用于去除蛋白质的任何聚合分数。仔细的 pH 调整和添加核苷酸的质量是至关重要的, 因为添加非调整或部分退化核苷酸到蛋白质储存或自组织化验足以消除蛋白质活动, 因此废除自组织。
其次是蛋白质质量, 膜质量是最关键的, 而不当的膜形成往往是导致缺陷自组织和 artefactual 表面结构的起源。
第一次执行该协议时, 通过在低摩尔百分比 (0.05%) 中添加标记脂类 (如 Atto-655-DOPE 或 DiI) 来标记支持的脂质双层是有帮助的。从而可以直接判断膜的性质和质量。使用酶, 可以评估膜的流动性。此外, 你可以直接评估 SLB 的洗涤质量, 因为如果洗掉了, 就会有太多的水泡, 没有液体膜, 或者根本没有膜。开放室的方法允许严格清洗膜, 因此也可以去除粘在 SLB 表面的水泡。获得液体和均匀支持的脂质双层的最关键的因素是表面的清洁和亲水性, 以及正确的尺寸和同质性.它可以帮助检查 SUV 的大小和大小分布使用动态光散射。对于狭窄的大小分布, 我们建议挤压的水泡, 而不是 sonicating 他们。其他清洁盖玻片的方法,例如,用结实的碱、基本的洗涤剂, 或者用清水冲洗后直接使用盖玻片, 可能会产生良好的效果, 这取决于应用和脂质混合物。
在这里提出的协议的前半部分,体外重建的平面支持脂质双层在开放室, 具有渲染的表面可接近光学镜, 如 TIRF 显微30, 酶分析6、单粒子跟踪34以及表面探针技术, 如原子力显微镜26。大齐次区域允许在定义的浓度下进行更好的统计。此外, 开放室方法允许精确控制蛋白质浓度和快速和简单的添加进一步的成分, 从而允许滴定蛋白质浓度在一个单一的房间20。通过添加其他细菌 divisome 成分, 如 FtsZ22、35、ZipA22或嵌合蛋白 FtsZ-YFP-MTS10、35, 也可扩大该检测。
其他团体也采取了类似的方法来重组最小系统的体外, 但使用流动细胞, 而不是开放室17,18,19。流动细胞有一定的优势, 特别是当一个完全封闭的3D 环境需要18, 流量17,19,23或膜组成23对 MinCDE 模式的影响是调查, 或如果蛋白质模式是要观察在蛋白质限制条件下19。然而, 局部控制分子浓度是比较困难的。蛋白质组分, 特别是头脑, 强烈束缚到他们第一次遇见的膜18,19。根据我们的经验, 这些蛋白质经常表现出对油管、入口、注射器和其他微流控部件的非特异约束。因此, 局部蛋白质浓度不同于输入浓度18 , 并且在流动细胞的长度上也有变化, 导致不同的蛋白质模式在入口和出口之间的膜, 如其他19所观察到的。
该议定书的后半部分提出, 在棒状显微组织中重新使用开放室方法的体外重建在脂质双层覆盖的图案支架上, 允许简单模拟体内蛋白行为。即使对蛋白质浓度的精确控制由于缓冲去除而丢失。请注意, 由于 MinDE 的波长比体内的一级大, 而在体外, 棒状 microcompartments 也大约是一级大 (10 x 30 µm), 而不是棒状大肠杆菌细胞。
总的来说, 该协议允许精确控制所有条件, 包括蛋白质浓度, 缓冲成分和膜性能。使用3D 结构化支持, 可以在空间约束下研究反应, 模仿体内行为而不需要复杂的微流体设备。

时空模式是必不可少的性质, 调节复杂的任务, 在多细胞和细胞水平, 从形态发生到调节细胞分裂1,2。反应扩散系统在建立这些模式方面起着重要作用, 但仍未得到很好的理解。反应扩散系统和最佳的特征的生物系统的一个典型例子到目前为止是大肠杆菌 MinCDE 系统3,4,5,6,7。在大肠杆菌中, MinCDE 系统从细胞极到细胞极振荡, 以确定细胞的中间为未来的分裂部位。该系统是基于 atp 的思想, atp 活化蛋白矿, 和膜作为一个空间反应矩阵8。MinC 不是模式形成机制的一部分, 而是实际的功能剂: 主要 divisome 蛋白 FtsZ5、6的抑制剂。MinC 结合到头脑, 因此跟随振荡, 导致平均蛋白质浓度梯度最大在细胞两极和极小的在细胞中间, 只允许 FtsZ 到聚合在 midcell9,10.MinCDE 系统是沃克大家庭的一部分, ATPases 是细菌2中时空组织的关键, 用于定位和运输蛋白质配合物11和质粒12以及调节细胞分裂13和染色体分离14。因此, MinCDE 反应扩散系统不仅代表了原型反应扩散系统, 而且由于其对细菌时空组织的相关性引起了人们的关注。
MinCDE 系统在体内的详细功能研究是复杂的, 因为蛋白质的操纵和基因的缺失通常会导致细胞分裂缺陷。此外, 改变膜的组成或细胞质的性质在体内是非常具有挑战性的15,16。在细胞的复杂环境中, 对系统和影响因素的改变很难解释, 更重要的是, 如果它在细胞分裂等基本功能中受到干扰。因此, 我们和其他人已经转向了一种体外重建方法, 将系统降低到其核心成分: 心, 矿, ATP 作为能量源, 以及支持的脂质双层作为反应基质6,17,18. 这种自下而上的方法允许在不复杂的活细胞的情况下, 详细探讨自我组织的机制。蛋白质形成旅行表面波浪6和其他种类样式17,19在这些情况下, 虽然以波长通常是大小大于在体内。使用开放室有助于精确控制影响模式形成的所有方面: 蛋白质浓度6, 蛋白质性质20, 膜成分10, 缓冲成分, ATP 浓度6、以及其他因素的增加, 如拥挤剂21等 divisome 蛋白22。相比之下, 在18、19、23流细胞中 MinCDE 系统的体外重建可以用来探测流动17、23、蛋白质的影响。限制条件19, 膜组成19和全3D 限制18的蛋白质模式, 但使精确控制蛋白质/成分浓度和顺序成分加法更复杂。
利用这个开放室, 我们还对平面脂质双层的支持进行了研究, 通过这种方法可以探讨几何边界对模式形成21的影响, 这种现象最近也在体内使用细菌模压成微结构7。我们还利用这一方法研究了在矿井中定义的突变是如何影响系统20的模式形成的。此外, 还采用了同样的基本化验格式来研究如何通过光照控制模式形成, 将偶氮苯交联的矿肽引入到检测中, 并用 TIRF 显微镜24进行成像。
我们发现, 为了复制体外观察体内的 MinDE 模式形成,禁闭是关键。使用棒状 microcompartments , 尺寸调整到 MinDE体外的较长 ( 10 x 30 µm ) , 用支持的脂质双层进行包覆 , 使 MinDE 极 - 极振荡和蛋白质梯度形成的重建10,25。在这项试验中, 所支持的脂质双层被沉积在一个有图案的, 包含数个百种复制品的棒状 microcompartments, 在顶端保持开放。由此, 反应可以建立在一个开放的房间, 随后缓冲区被降低到 microcompartments 的边缘, 从而限制了对小体积的反应。虽然这些隔间有一个空气缓冲接口在一侧, 因此并不代表一个完整的3D 隔离膜, 蛋白质动力学模拟在体内振荡10,25。与全3D 隔离, 这表明非常相似的结果18, 开放的显微结构测试是相对简单, 易于处理, 也可以由实验室进行, 没有配备专门的微流体设备和干净的房间设施。
在这里, 我们提出了一个实验性的协议, 以重建 MinCDE 模式形成的支持脂质双层的体外使用一个开放的房间, 允许控制所有组件和容易访问的光学显微镜, 并与小修改, 也适用于表面探针技术26。在平板支持的脂质双层的旁边, 我们还展示了如何使用简单的图案支持的脂质双层在棒状的新的微结构上获得蛋白质约束。这些化验, 虽然对 MinCDE 系统进行了优化, 但也可以转移到其他与膜类似的蛋白质系统, 如 FtsZ27或最小肌动蛋白皮质28。

<table>
	<tbody>
		<tr>
			<td colspan="4">Reagents</td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPG</td>
			<td>Avanti Polar Lipids</td>
			<td>840475</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli polar lipid extract</td>
			<td>Avanti Polar Lipids</td>
			<td>100600</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt trihydrate</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-diphosphate monopotassium salt dihydrate</td>
			<td>Sigma</td>
			<td>A5285-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>27810.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma Aldrich</td>
			<td>T1503-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Roth</td>
			<td>9277.1</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP-HCl</td>
			<td>Termo Fisher Scientific</td>
			<td>20491</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetate</td>
			<td>Merck Millipore</td>
			<td>1.08418.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric Acid 98%</td>
			<td>Applichem</td>
			<td>173163.1611</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 50%</td>
			<td>Applichem</td>
			<td>147064.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Biomol</td>
			<td>05288.1</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>Merck</td>
			<td>102950</td>
			<td>Uvasol</td>
		</tr>
		<tr>
			<td>Glycerol 86%</td>
			<td>Roth</td>
			<td>4043.1</td>
			<td></td>
		</tr>
		<tr>
			<td>TB medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Roth</td>
			<td>2316.x</td>
			<td></td>
		</tr>
		<tr>
			<td>Atto-655-DOPE</td>
			<td>Atto Tec</td>
			<td>AD 655-161</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS base</td>
			<td>Dow Corning Corporation</td>
			<td></td>
			<td>SYLGARD 184</td>
		</tr>
		<tr>
			<td>PDMS crosslinker</td>
			<td>Dow Corning Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Materials</td>
		</tr>
		<tr>
			<td>UV Glue</td>
			<td>Norland Products</td>
			<td>6801</td>
			<td>#68 and #63 both work well</td>
		</tr>
		<tr>
			<td>Coverslips #1.5 24x24 mm</td>
			<td>Menzel Gl&#228;ser</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips #1 24x24 mm</td>
			<td>Menzel Gl&#228;ser</td>
			<td></td>
			<td>used only for PDMS microstructures</td>
		</tr>
		<tr>
			<td>0.5 ml reaction tube</td>
			<td>Eppendorf</td>
			<td>&#160;0030123301</td>
			<td></td>
		</tr>
		<tr>
			<td>culture flask 2L</td>
			<td>Corning</td>
			<td>e.g. 734-1905</td>
			<td></td>
		</tr>
		<tr>
			<td>His-Trap HP</td>
			<td>GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfiltration column: HiLoad Superdex 75 PG or 200 PG</td>
			<td>GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Econo-Pac 10DG desalting column prepacked column</td>
			<td>Biorad</td>
			<td>7322010</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis device: Slide-A-Lyzer Dialysis Cassettes, 3.5K MWCO, 0.1 - 0.5 mL or 0.5-3 mL</td>
			<td>Termo Fisher Scientific</td>
			<td>66333 or 66330</td>
			<td></td>
		</tr>
		<tr>
			<td>razor blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Instruments</td>
		</tr>
		<tr>
			<td>ultrapure water: Milli-Q Type 1 Ultrapure Water Systems</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>automated protein purification system: &#196;kta Pure</td>
			<td>GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bath sonicator</td>
			<td>Branson</td>
			<td></td>
			<td>e.g. Model 1510</td>
		</tr>
		<tr>
			<td>ARE-250 mixer</td>
			<td>Thinky Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner Zepto</td>
			<td>Diener electronic</td>
			<td></td>
			<td>use oxygen as process gas</td>
		</tr>
		<tr>
			<td>positive displacement pipettes</td>
			<td>Brand</td>
			<td></td>
			<td>Transferpettor models with glass tips</td>
		</tr>
		<tr>
			<td>LSM780 confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Fitted with Zeiss C-Apochromat 40X/1.20 water-immersion objective</td>
		</tr>
		<tr>
			<td colspan="4">Plasmids</td>
		</tr>
		<tr>
			<td>pET28a-His-MinD_MinE</td>
			<td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td>
			<td></td>
			<td>plasmid encoding His-MinD and non-tagged MinE to improve yield</td>
		</tr>
		<tr>
			<td>pET28a-His-MinE</td>
			<td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td>
			<td></td>
			<td>plasmid encoding His-MinE</td>
		</tr>
		<tr>
			<td>pET28a-His-EGFP-MinD</td>
			<td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td>
			<td></td>
			<td>plasmid encoding His-EGFP-MinD</td>
		</tr>
		<tr>
			<td>pET28a-His-mRuby3-MinD</td>
			<td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td>
			<td></td>
			<td>plasmid encoding His-mRuby3-MinD</td>
		</tr>
		<tr>
			<td>pET28a-His- MinC</td>
			<td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td>
			<td></td>
			<td>plasmid encoding His-MinC</td>
		</tr>
	</tbody>
</table>

in vitro reconstitution of self-organizing protein patterns on supported lipid bilayers

细胞的基本时空组织的许多方面都受反应扩散型系统的控制。对这种系统进行体外重建, 可以对其在体内不可行的基本机制进行详细研究。在此, 我们为大肠杆菌 MinCDE 系统的体外重建提供了一种协议, 即在细胞中间放置细胞分裂隔膜。该试验的目的是只提供自组织所需的成分, 即膜, 两个蛋白质的头脑和矿山和能源的形式, ATP。因此, 我们在盖玻片上制造一个开放的反应室, 形成一个支持的脂双层。该室的开放式设计, 使脂质双层和控制操作的散装内容的最佳准备。这两种蛋白质, 头脑和我的, 以及 ATP, 然后添加到大容量的膜上。成像是可能的许多光学镜, 因为设计支持共焦, 宽领域和 TIRF 显微镜一样。在该协议的变化, 脂双层是形成了一个图案的支持, 在细胞形的, 而不是玻璃。将体积溶液降低到这些隔间的边缘将反应放在一个较小的车厢内, 并提供允许模仿体内振荡行为的边界。结合起来, 我们描述了重建 MinCDE 系统的协议, 既具有空间限制, 又允许研究人员精确控制影响模式形成的各个方面, 如浓度范围和其他因素的增加。或蛋白质, 并系统地增加系统复杂度相对简单的实验设置。

蛋白质纯化后, 我们的协议应产生纯度最低的蛋白质。作为参考,图 1提供了一个荧光的想法, 标签的头脑, 地雷和 MinC。图 2描述了在非图案支持的脂质双层上执行 MinDE 自组织检测过程的各个步骤。使用此协议, 可以在整个会议厅中观察到常规的 MinDE 行表面波 (图 3)。在腔内波长可能略有变化, 但一般模式看起来相似。该腔的边缘不应用于定量比较, 因为在 UV 胶上形成的膜似乎有不同的性质比在玻璃表面 (见图 3C)。通过绘制沿传播方向的强度来分析行面波 (图 3B)。当头脑荧光高原相当快速从波浪的前缘, 然后急剧地减少在尾随边缘时, 矿荧光从头脑波浪的开始几乎线性地增加并且到达它的最大在头脑以后在后面边缘, 在那里它明显下降6。
在蛋白质质量的旁边, 支持的脂质双层的质量对 MinCDE 的定期自组织是最关键的。一方面, 如果膜过度清洗或底层表面被清洗, 因而充电过强, 孔可以形成在膜 (图 6A, 顶部)。另一方面, 如果膜未正确清洗或底层表面未清洗/hydrophilized, 泡膜会粘在细胞膜上或膜的流动性将受到损害 (图 6A, 底部)。即使不像在直接通过标记脂质观察膜时那样明显, 这些问题也可以从心智荧光信号中检测出来, 因为模式不是规则的, 而最大值中的荧光不是同源的, 而是包含 "孔" 或如图 6A的中间面板所示的明亮点。
对 MinDE 自组织的棒状聚硅烷微结构的过程概述在图 4。一些协议步骤不需要重复, 因为蛋白质和脂质可以被重用。像在非图案基板上, 该基底被清洗和 hydrophilized (通过等离子清洗), 一个支持的脂质双层形成在该公司和自组织试验建立在200µL 的数量。为了检查一个适当的膜已经形成和 MinDE 自组织在膜上, 分庭是成像。当形成适当的膜时, MinDE 在微结构的表面形成规则的行面波, 并且在微结构的底部自组织, 因为波浪可以自由地移动整个膜覆盖表面 (图 5A)。去除缓冲区后, 隔间的表面不应显示任何传播的 MinDE 模式 (图 5B), 因为它应该是完全干燥的。如果 MinDE 模式仍在移动, 则需要删除更多的缓冲区。这些蛋白质现在被膜覆盖的 microcompartments 和上层界面上的空气 (图 5C) 限制在棒状的, 它们将自行组织。在这种情况下, 这两种蛋白质可以执行极到极点的振荡, 如图 5D所示。作为头脑和矿的一小部分总是膜束缚的, 也在缓冲去除期间, 在缓冲去除以后的浓度不与输入浓度相媲美。由于这种影响, 浓度也在不同的微观组织在相同的盖玻片, 因为它们取决于模式的位置之前, 缓冲区删除。硅片的生产或硅晶片的注塑可能导致不完全的显微组织, 不能用于分析 (图 6B)。此外, 由于缓冲去除微结构可能在过程中干燥, 因此, 应排除在进一步分析 (图 6B)。因此, 只有一小部分的显微组织在一个房间显示所期望的极点到极点的振荡。为了分析显微组织中的蛋白质动力学, 可以通过在整个结构上绘制一个选择来获得 kymograph (图 5E)。当 MinCDE 振荡从极到极点, MinC 和头脑将显示一个时间平均浓度梯度, 最大浓度在室极和最低浓度在车厢中间 (图 5F)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58139/58139fig1.jpg"> 图 1: 显示蛋白质纯化最终产物的 SDS 页.他的头脑 (33.3 kDa), 他的 eGFP 头脑 (60.1 kDa), His-mRuby3-MinD (59.9 kDa), 他的矿 (13.9 kDa) 和他的 MinC (28.3 kDa) 按顺序显示。<a href="https://www.jove.com/files/ftp_upload/58139/58139fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58139/58139fig2.jpg"> 图 2: 显示非图案支持脂质双层的自组织协议的各个步骤和时间的流程图 (步骤 1-6).虚线框表示这两个选项之一可用于清洗。由圆圈标记的箭头指示可以在以后暂停和恢复协议。<a href="https://www.jove.com/files/ftp_upload/58139/58139fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58139/58139fig3.jpg"> 图 3: 共焦显微成像 MinDE 检测.A)规则最小螺旋, 可获得波传播速度、强度图和速度测量。使用的浓度: 0.6 µM (30% eGFP 心), 1.8 µM 他的地雷 (30% His-MinE-Alexa647) B)示例中标记为. C的区域的规范化强度图. 整个化验室概述 (刻度条: 1 毫米, 相同的蛋白质浓度为以上)。可以观察螺旋旋转方向和目标模式。放大的区域显示了在 UV 胶水上波浪图案的不同。<a href="https://www.jove.com/files/ftp_upload/58139/58139fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58139/58139fig4.jpg"> 图 4: 显示了杆形微结构中自组织协议的各个步骤和时间的流程图 (步骤1-5、7、8).灰色框表示可在不带图案支持的脂质双层的协议中重用产品的步骤。由圆圈标记的箭头指示可以在以后暂停和恢复协议。<a href="https://www.jove.com/files/ftp_upload/58139/58139fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58139/58139fig5.jpg"> 图 5: 杆型 microcompartments 中 MinDE 模式形成的代表性结果。A) MinDE 自组织在表面上形成行面波 (1 µM 心 (30% EGFP), 2 µM 矿和2.5 毫米 ATP)。B)在缓冲液降至微结构高度后, 蛋白质自组织在 microcompartments 之间的平面表面停止。C)一杆形 microcompartment 的示意图。D)缓冲区去除后 MinDE 极-极振荡的代表性图像。E) Kymograph 在 D 中显示的突出线沿线的振荡。F)在 D 中显示的时间序列的平均荧光强度的图像和剖面图清楚地显示了在 microcompartment 极和中间间隔最小的蛋白质梯度。<a href="https://www.jove.com/files/ftp_upload/58139/58139fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58139/58139fig6.jpg"> 图 6: 阴性实验结果的例子.A)过度冲洗的膜堆积孔, 而次优囊泡制剂和脂质成分导致囊泡。两个中心面板显示了两个问题的组合, 以及它们在观测最小振荡时如何变得可见。膜被贴上 0.05% Atto655-DOPE 的标签。(刻度条:50 µm)B)顶部面板: 干燥 microcompartments 可能是由于太多的缓冲区删除或当缓冲区蒸发随着时间的推移。底板: 在硅片生产或塑料成型过程中可形成不完整的隔间。(刻度条:30 µm)<a href="https://www.jove.com/files/ftp_upload/58139/58139fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充文件 1:质粒图谱为他的头脑。<a href="https://www.jove.com/files/ftp_upload/58139/01_pET28a-MinD-MinE.gb%20" target="_blank">请单击此处下载此文件.</a>
补充文件 2:质粒图谱为他的 EGFP 头脑。<a href="https://www.jove.com/files/ftp_upload/58139/02_pET28a-EGFP-MinD.gb" target="_blank">请单击此处下载此文件.</a>
补充文件 3:His-mRuby3-MinD 质粒图谱。<a href="https://www.jove.com/files/ftp_upload/58139/03_pET28a-mRuby3-MinD.gb" target="_blank">请单击此处下载此文件.</a>
补充文件 4:质粒图为他的地雷。<a href="https://www.jove.com/files/ftp_upload/58139/04_pET28a-MinE.gb" target="_blank">请单击此处下载此文件.</a>
补充文件 5:MinC 的质粒图谱。<a href="https://www.jove.com/files/ftp_upload/58139/05_pET28a-MinC.gb" target="_blank">请单击此处下载此文件.</a>
补充文件 6:棒形 microcompartments 硅片的 CAD 文件。<a href="https://www.jove.com/files/ftp_upload/58139/JoVe_wafer_rod-like-structures_3x3.dwg" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers at JoVE.com

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

我们提供了一个协议,在体外自我组织的精神和矿山的研究, 在支持的脂质双层在开放室。此外, 我们还描述了如何将该检测包裹在脂质包覆的 microcompartments 中, 通过反应约束模拟体内条件。

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

in-vitro-reconstitution-self-organizing-protein-patterns-on-supported

Research

JoVE Journal

Biochemistry

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

12.1K 次观看.  Max Planck Institute of Biochemistry. 我们提供了一个协议,在体外自我组织的精神和矿山的研究, 在支持的脂质双层在开放室。此外, 我们还描述了如何将该检测包裹在脂质包覆的 microcompartments 中, 通过反应约束模拟体内条件。

视频: In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

体外F-肌动蛋白在早期体的聚合

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

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 ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an enormous amount of spectra, setting challenges for quality control of the mass spectrometry spectra. To reduce the false positives, false discovery rate (FDR) quality control is necessary. Recently, we developed a software for FDR control of untargeted metabolome identification that is based on a Target-Decoy strategy named XY-Meta. Here, we demonstrated a complete analysis pipeline that integrates XY-Meta and metaX together. This protocol shows how to use XY-meta to generate a decoy database from an existing reference database and perform FDR control using the Target-Decoy strategy for large-scale metabolome identification on an open-access dataset. The differential analysis and metabolites annotation were performed after running metaX for metabolites peaks detection and quantitation. In order to help more researchers, we also developed a user-friendly cloud-based analysis platform for these analyses, without the need for bioinformatics skills or any computer languages.

We constructed an untargeted metabolomic workflow that integrated XY-Meta and metaX together. In this protocol, we displayed how to use XY-Meta to generate a decoy spectral library from open access spectra reference, and then performed FDR control and used the metaX to quantitate the metabolites after identifying the metabolomics spectra.

基于FDR对照的非靶向代谢组的鉴定和定量集成工作流程

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an ...