Name: identification of plant ice-binding proteins through assessment of ice-recrystallization inhibition and isolation using ice-affinity purification
Uploaded: 2017-05-05T13:00:00.000+00:00
Duration: 9 min 43 s
Description: Watch this Scientific Journal Video about Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification at JoVE.com

这项工作是由NSERC（加拿大）赠款，以资助VKW。

1.啪的装置设置<ol><li>填与乙二醇（50％V / V的水溶液）的温度可编程循环水浴中。 注:绿色汽车乙二醇可以使用。 </li><li>为了组装外部腔室，使用绝缘塑料管的水浴附加到双壁玻璃碗。绝缘玻璃碗聚苯乙烯泡沫并用塑料皮氏培养皿盖上盖子。切割在聚苯乙烯室的底部3-4英寸的孔，使得光源可通过玻璃腔室中可以看出。 </li><li>安装在解剖显微镜外室，安装有摄像机端口和与10个目镜一个4X极化物镜。放置偏光膜的附加件在所述外部腔室的底部。 </li><li>夹持1-1.5米管（〜5cm直径）到大聚苯乙烯容器内的铁架台和地点，与位于下方管在铁架台的基座的冷却块沿。 </li></ol> 2.天然蛋白提取物的制备用于IRI分析<ol><li>为了诱导IBPS在冷冻耐受草或其它冷冻耐受性的植物物种，冷驯化植物中表达为最多1周，在4℃，低光照条件下（6小时光照/ 18小时黑暗）下。这并不适用于从已经暴露在低温条件下驯化期间实地收集的样本。 </li><li>通过在杆的基部切割得到约0.1g叶组织，然后将在1.5mL管。立刻闪在-80℃下直至使用冷冻在液氮中并保存样品。 </li><li>为了裂解细胞，研磨组织样品放入使用液氮下用研钵和研杵细粉。确保液体氮不均质化工序蒸发。 </li><li>立即置于冰上研磨样品，并允许液氮完全蒸发。加入1毫升的天然蛋白质extracti的上含10mM的Tris-HCl，25mM的NaCl的（pH 7.5）中，与在使用前立即添加了两个无EDTA的蛋白酶抑制剂片剂缓冲器。移液器混合;不要振荡。 注:其他添加剂可能需要被使用，如果所述裂解物含有次级代谢产物（见讨论）。 </li><li>轻轻摇动，在4℃过夜的样品（18-24小时）。进行在4℃下的其余步骤。 </li><li>通过在〜16000×g下离心样品5分钟，除去细胞碎片。保留的可溶性级分，离心额外的5分钟，如果细胞碎片仍然存在。澄清的样品可以储存在-20℃直至使用。 </li></ol> 3.片分析预实验设置<ol><li>倒100毫升己烷到外部腔室设置与该偏光膜，并添加5-6干燥珠粒，以防止湿气积聚在滑动。真空润滑脂少量添加到所述板覆盖所述浴和扭曲，以确保紧密的密封，并防止己烷的蒸发。 </l我&gt; <li>在开始实验之前冷却℃，-6℃使用可编程水浴大约2-3小时之间-4己烷浴中。请务必检查正己烷的温度之前开始实验温度计。 </li><li>放置冷却块在正下方的塑料管中的聚苯乙烯盒，并用干冰完全覆盖之前，开始实验40分钟。 </li><li>在开始测试之前，确保管处于水平位置。为了确定其中冷却块中的样本将下降，第一测试在与水的程序和指示在其中样品与标记下降（如下所述）。 </li><li>从冷块的顶部除去干冰，并放置在已被确定与水滴标记玻璃显微镜盖玻片。 </li><li>立即开始测定前，打开光源上并刮去已形成在玻璃腔室的底部的任何冰。避免保持光源上，除非冰晶被可视化，以防止加热己烷。 </li></ol> 4.导电的压片分析<ol><li>浸一次性尖端固定到在浸油的自动吸管（1-20微升），用纸巾除去任何多余的油。这一步将允许样品下降，而不是坚持移液管的外侧。 注:从吸管去除多余油脂关键是要确保油滴不会形成冰晶体，使得可视化困难。 </li><li>移液管10μL样品和地点移液管直接在释放样品之前塑料管。当样品撞击玻璃盖玻片，创造冰晶的单层一个独立的"图示"的声音应该被听到。 </li><li>快速并小心地取出从使用镊子和发生在该偏振膜的顶部上的己烷浴中冷块中的显微镜载玻片上。 </li><li>在显微镜下看，把显微镜载玻片进入视和广告领域只是物镜，使得交叉极化允许高对比度冰晶的可视化。调整放大倍数40倍。 </li><li>将相机安装到显微镜和调整到"宏"的设置，以允许冰晶体的清晰的可视化，并捕获图像。 注:等待长达1个小时，使冰晶生长可以给更清晰的画面。 </li><li>重复步骤4.1-4.5所有样品。通常，放置最多6个滑动与在己烷腔室中的不同的样品。 </li><li>关闭光源并放置在己烷浴塑料板，确保紧密的密封。允许冰晶退火过夜（12-24小时，保持一个特定测定内一致的退火期间）。在温育之后，如上文所描述的冰膜的捕获图像。 注:某些样本可能比其他人更快地再结晶。如果12个小时后没有再结晶是明显的，允许晶体退火达24小时，以确保没有recrystalli矩阵特殊积会发生。 </li></ol> 5.片分析数据分析<ol><li>上传照片到电脑前和退火后直接比较冰晶的大小。用冰再结晶活性的样品不会生长，而没有无IRI活动样本将重结晶形成明显更大的冰晶。光干扰会使大量冰晶出现在稍微不同的色调，因此很容易看到。 </li></ol> 6.冰亲和力净化设备安装<ol><li>填与乙二醇的温度可编程循环水浴中。绿色汽车乙二醇可以使用。 </li><li>浴连接到使用绝缘塑料管15的中空，黄铜探针。 </li><li>构造一个聚苯乙烯容器，其中一个150毫升烧杯中，可紧贴地配合。创建用于与中心为黄铜​​指形冷冻器中的孔的容器的盖子。 </li></ol> 7。样品的制备用于冰亲和纯化<ol><li>冷驯化植物组织中第2.1节所描述的。 </li><li>收集〜100-150克在20个毫升管中的地上生物量（步骤2.3）的并立即闪烁冻结。样品可以保持在使用前-80℃。 </li><li>如在章节2.3-2.5描述制备样品。使用1:1倍的比例（mg组织:天然蛋白质提取缓冲液中的溶液），用于叶组织的再悬浮。使用额外的蛋白酶抑制剂药片通过内源蛋白酶，以避免IBPS的降解。 注意:如果所述组织样品是过于集中，调整比率为1:2或1:3（mg组织的:天然蛋白质提取缓冲液中的溶液）。 </li><li>以去除细胞碎片，通过2层干酪包布的，分3次筛裂解物。沉淀通过离心碎片以30,000×g于4℃下40分钟。 </li><li>增加样品的使用天然蛋白质提取缓冲液120毫升的体积。立即使用裂解液冰亲和纯化，以避免冰结合活性的任何损失。 </li></ol> 8.导电冰亲和纯化<ol><li>在纯化前，设置可编程水浴从-0.5℃至冷却在-0.04℃/ h的速率至-3℃。 注:冷却速度可根据样品的初始体积进行调整。 </li><li>冷却冰手指至-0.5℃，并随后将其浸没到含有冷蒸馏水50mL试管。添加一些冰块，以促进冰成核和允许冰的薄层，以形成在手指上。这个过程通常需要20分钟到30之间。 注:在冰面上涂覆的手指的任何团块应该用戴着手套的手在样品放置之前被平滑化。如果冰层不会在-0.5℃下形成，将温度降低到-0.75℃。 </li><li>地方样品中的150姆·格拉斯烧杯中含有小的磁力搅拌棒放入在磁力搅拌器上的顶部上的聚苯乙烯容器。确保冰手指居中并降低至少一半的方式进入液体样品。密封容器。 </li><li>允许程序大约运行了两天，检查每24小时。一旦样品的50％被冻结，停止该程序。 IBPS的纳入冰半球会导致"冰蚀刻"。 注:实验纯化可使用较大的样品体积随着时间的长度在该程序运行相应的调整来进行。 </li><li>为了除去冰半球，温探测到4℃，冲洗半球用蒸馏水以除去未结合的蛋白质，然后在4℃下解冻。 注意:大多数IBPS不是水和适当的缓冲液稳定的（ 即 50毫摩尔Tris-HCL，pH为8或50mM碳酸氢铵，pH 8）中将需要在解冻过程中定期加入（约1毫升/ 2小时） 。冰半球可以包裹在铝箔和解冻之前储存在-20℃。 </li><li>如果冰半球还含有小号颜料，或以增加样品纯化，重复步骤8.1-8.5 2-3次。 注意:虽然IBP的浓度可以更低以下多轮的纯化，纯度大于屈服如果纯化的样品将使用MS分析更有价值。如果有足够的材料是可用的，建议三轮冰 - 亲和纯化的。 </li></ol> 9. IBPS的鉴定<ol><li>由于IBPS跟随冰半球的解冻包含在一个大的体积，浓缩的样品。集中使用离心浓缩管与3000沓分子量截止，以避免小蛋白质的损失含有约100 g组织至〜1-3毫升样品。 </li><li>在发送之前样品的MS，确定使用蛋白定量测定法中的蛋白质浓度，如BCA或Bradford测定。通过凝胶电泳例如SDS-PAGE估计的样品的纯度。之前测试IRI活动的纯化蛋白质瑟nding为MS，以确保活性蛋白已经通过纯化和浓缩步骤保留。 </li><li>直接使用浓缩的样品用于质谱分析10。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+evolution+of+a+cold+stress+associated+gene+family+in+cold+tolerant+grasses.">Tracking the evolution of a cold stress associated gene family in cold tolerant grasses.</a> BMC Evol. Biol. 8 (245), (2008).</li><li>Middleton, A. J., Brown, A. M., Davies, P. L., Walker, V. K. <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+the+ice-binding+face+of+a+plant+antifreeze+protein.">Identification of the ice-binding face of a plant antifreeze protein.</a> FEBS Lett. 583 (4), 815-819 (2009).</li></ol>

作者什么都没有透露。

对于IBPS成功的分析和纯化，了解一些这些蛋白质的温度敏感性是非常重要的。某些植物IBPS在温度高于0℃变得不稳定，导致解折叠，沉淀和不活动。为了获得活性IBPS，通常关键的是植物在冷室（〜4℃）处理，并在实验过程中样品保持在冰上。另一个因素考虑使用全细胞裂解物的粗时是通过内源蛋白酶的蛋白质的降解。在植物中，IBPS的表达一般是低的，并且因此在蛋白质产量的损失可能导致材料?...

冰-结合蛋白（IBPS）是已在许多生物包括植物1，昆虫2，鱼3，和微生物4的被发现的保护蛋白质的不同的家族。这些蛋白质的关键特征是其独特的特异且高效地吸附到冰晶，修改他们的生长的能力。 IBPS具有若干记录性质，具有两个最充分表征的是热滞后（TH）和冰再结晶抑制（IRI）。 TH活性在以冷冻不耐动物产生IBPS更容易地观察到。此活性导致有机体循环或间质液的凝固点降低，以防止冻结。相反，在冷冻耐受性生物体，这将不可避免地冻结在零度以下的温度，IBPS似乎具有低TH活性。尽管低TH活性，高的IRI活动限制冰哭飞虱生长通常与这些蛋白质观察到。对于冷冻宽容有机体，这IRI活动可能有助于保护细胞免受冰的细胞外车厢的不受控制的生长。 的"床垫键"模型可以用于描述通过该IBPS防止冰晶5的生长机制。在这种模式下，特别IBPS吸附于在时间间隔，使得水分子可以仅与结合IBPS之间的空间中生长冰晶格掺入冰晶体的表面。这就形成了一个曲率，使额外的水分子的掺入不利的，可以由吉布斯·汤姆逊效应6描述的事件。锚定笼形水域假说提供了IBPS的特异性结合到冰晶体表面上的机构，从而电荷的残基的存在，特别是定位在蛋白冰结合位点上，结果在呼肠孤水分子的rganization所以它们匹配冰晶格7的一个或多个平面。 TH活性可通过测量熔化之间的差和在IBP的存在冻结单个冰晶的温度进行定量。而TH活性评价IBPS，通过植物IBPS（通常仅一个度的一小部分）中产生的低TH间隙的活性的广泛接受的方法通常需要较高的蛋白质浓度，专门的设备和操作员的经验。虽然非IBPS可限制冰晶生长，它是由所有IBPS因此测试对于IRI活动共享的属性被用于IBPS存在一个有效的初始屏幕上，特别是对那些具有低TH活性。用于测试该活动的方法是被称为"压片分析"，由此蛋白质样品快速冷冻，以产生的小冰晶，其在一段时间，以确定是否观察到单层冰晶生长受到限制。不同于用于筛选IBPS的存在的源组织样品的其它方法，该技术适用于在10-100纳克的范围内的低的蛋白质浓度，使用容易制造的设备，并且产生快速且容易地解释的数据。然而，强调的是，该测定提供了一个初始屏幕应遵循由TH的确定和冰晶成形IBPS是重要的。 天然蛋白质的分离是具有挑战性的，通常需要关于感兴趣的蛋白质的结构和生化特性的信息。 IBPS的冰的亲和力允许使用冰作为用于纯化目的的基板这些蛋白质的分离。由于多数分子被冰晶生长，在一个高度纯化样品中的IBP样品的存在导致冰 - 半球的生长缓慢，不含大quantit期间提前冰水边界的推污染的蛋白质和溶质的IES。该方法已被用于从昆虫8，9，10，11的细菌，鱼12和植物13,14识别IBPS。此外，通过这种方法实现的IBP-富集的级分也可用于下游生化分析。本文概述IBPS在植物通过IBPS的诱导和提取的识别，IRI活性的分析，以证实蛋白质的存在，并使用冰亲和纯化的蛋白质的分离。

<table><tbody><tr><td>1.5 mL microcentrifugetubes</td><td>Fisher</td><td>05-408-129</td><td></td></tr><tr><td>Adjustable lab jack</td><td>Fisher</td><td>S63080</td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Desaga MC2</td><td></td><td></td></tr><tr><td>Brass probe</td><td>Custom built</td><td></td><td></td></tr><tr><td>Camera/camera port</td><td>Canon</td><td></td><td>Canon Power Shot SX110 digitial camera; custom built microscope port</td></tr><tr><td>Cheesecloth</td><td>Purewipe/Fisher Scientific</td><td>06-665-25A</td><td></td></tr><tr><td>Concentration tubes (0.5 mL)</td><td>EMD Millipore</td><td>UFC501008</td><td></td></tr><tr><td>Concentration tubes (15 mL)</td><td>EMD Millipore</td><td>UFC900308</td><td></td></tr><tr><td>Conical tubes (50 mL)</td><td>Thermo Fisher</td><td>AM12502</td><td></td></tr><tr><td>Cooling block</td><td>VWR</td><td>13259</td><td>Use a metal heating block</td></tr><tr><td>Dehumidifier</td><td>Whirlpool</td><td></td><td>50 pint Energy Star dehumidifier; purchase from local supplier</td></tr><tr><td>Dessciation beads</td><td>t.h.e. Dessicant/VWR</td><td>EM-DX0017-2</td><td>6-8 mesh size; 100% indicating</td></tr><tr><td>Dissection microscope</td><td>Olympus Tokyo</td><td></td><td></td></tr><tr><td>Double walled glass bowl</td><td>Generic</td><td></td><td>Purchase from local lab glassware supplier</td></tr><tr><td>Dry ice</td><td>Generic</td><td></td><td>Use local supplier; hazardous&amp;nbsp;</td></tr><tr><td>EDTA-protease inhibitor tablets</td><td>Sigma Aldrich</td><td>11836170001</td><td>Roche cOmplete mini</td></tr><tr><td>Ethylene glycol</td><td>Generic</td><td></td><td>Green automotive ethylene glycol can be purchased from any local hardware store (&lt;em&gt;i.e.&lt;/em&gt; Home Depot)</td></tr><tr><td>Hexane</td><td>Sigma Aldrich</td><td>296090</td><td>Anhydrous, 95%; hazardous</td></tr><tr><td>Immersion oil</td><td>Sigma Aldrich</td><td>56822</td><td></td></tr><tr><td>JA10/20 centrifuge</td><td>Beckman</td><td></td><td></td></tr><tr><td>Large plastic Petri dish</td><td>Generic</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td>Generic</td><td></td><td>Use local supplier; hazardous&amp;nbsp;</td></tr><tr><td>Magnetic stir plate</td><td>Hanna Instruments</td><td>HI190M</td><td></td></tr><tr><td>Microscope cover slides</td><td>Fisher</td><td>12-542A</td><td></td></tr><tr><td>Plastic tube</td><td>Generic</td><td></td><td>Purchase PVC pipe from local hardware store</td></tr><tr><td>Polarized film</td><td>Edmund Optics</td><td>43-781</td><td></td></tr><tr><td>Polystyrene foam</td><td>Generic</td><td></td><td>Can be constructed from polystyrene shipping boxes</td></tr><tr><td>Poreclain mortar and pestle</td><td>Fisher</td><td>FB961</td><td></td></tr><tr><td>PVPP</td><td>Sigma Aldrich</td><td>77627</td><td>110 &amp;micro;m particle size</td></tr><tr><td>Retort Stand</td><td>Fisher</td><td>12-000</td><td></td></tr><tr><td>Small stir bar</td><td>Fisher</td><td>14-513-51</td><td></td></tr><tr><td>Temperature-programmable water bath</td><td>VWR</td><td>13271-118</td><td></td></tr><tr><td>Vacuum grease</td><td>Dow Corning/Sigma Aldrich</td><td>Z273554</td><td></td></tr><tr><td>Vinyl tubing</td><td>Generic</td><td></td><td></td></tr></tbody></table>

identification of plant ice-binding proteins through assessment of ice-recrystallization inhibition and isolation using ice-affinity purification

冰 - 结合蛋白（IBPS）属于家族，其通过暴露于零下温度某些生物体合成应激诱导的蛋白质。在植物中，当胞外冰晶生长发生冷冻损伤，导致质膜和可能的细胞死亡的破裂。 IBPS吸附到冰晶通过被称为冰再结晶抑制（IRI），从而减少细胞损伤的方法限制了进一步的增长。 IBPS还证明压下平衡熔点以下的溶液，被称为热滞后（TH）活性的特性的凝固点的能力。这些防护性能都提出了小说IBPS鉴定兴趣，因为它们在工业，医疗和农业应用的潜在用途。本文描述的植物的IBPS通过1识别）IBPS的植物组织中诱导和提取，2）提取用于IRI活性的筛选，以及3）的分离和PURIFIBP的使用。在通过低温暴露诱导IBP后，使用"splat测定"测试提取物的IRI活性，其允许使用标准光学显微镜观察冰晶生长。该测定需要低蛋白质浓度并产生快速获得并容易解释的结果，为冰结合活性提供初步筛选。然后可以通过利用IBPs吸附到冰上的特性，通过称为"冰亲和纯化"的技术将IBP从污染蛋白质中分离出来。使用从植物提取物收集的细胞裂解液，可以在黄铜探针上缓慢生长冰球。这将IBP融入多晶冰的晶体结构中。不需要IBP 的先验生物化学或结构学知识，这种方法可以恢复活性蛋白质。冰纯化的蛋白质级分可用于下游应用，包括pep的鉴定通过质谱法和天然蛋白质的生化分析潮序列。

为了便于设置， 图1和 图2被包括作为分别用于IRI分析和冰-亲和纯化，该设备的视觉表示。使用来自芥菜杂草收集有和没有IBPS提取IRI分析的结果在图3中被描绘。这些结果表明，从芥末杂草，这是不冷冻耐受收集提取物，无法限制冰晶生长由于缺乏IBPS本的。相比之下，小冰晶看到用含有从多?...

Watch this Scientific Journal Video about Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification at JoVE.com

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

本文概述冰结合蛋白的从冷冻耐受性植物通过冰再结晶抑制活性，并使用冰 - 亲和纯化天然IBPS的随后分离的评估鉴定。

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

identification-plant-ice-binding-proteins-through-assessment-ice

Research

JoVE Journal

Biochemistry

8.6K 次观看.  Queen's University. 本文概述冰结合蛋白的从冷冻耐受性植物通过冰再结晶抑制活性，并使用冰 - 亲和纯化天然IBPS的随后分离的评估鉴定。 

视频: Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

LabVIEW操作的新型纳升渗透压仪冰结合蛋白的调查

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

科研

化学

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

生物化学

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

高分辨率红外热成像（HRIT）的冰核和冰传播的植物研究中的应用

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

环境科学

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

毛细管电泳与亲和毛细管电泳对金属离子 AtHIRD11 固有紊乱与结合的分析

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the formation of single microscopic crystals and a suitable microscopy system coupled to the cold stage. The method described herein adds another important feature that includes exchanging solutions around ice and clathrate hydrate crystals. The described system comprises a combination of unique and home-developed instruments, including microfluidics, high-resolution cold stages, and fluorescence microscopy. The cold stage was designed for microfluidic devices and allows for the formation of micron-sized ice/hydrate crystals inside microfluidic channels and the exchange of solutions around them. The temperature resolution and stability of the cold stage is one millikelvin, which is crucial for controlling the growth of these small crystals. This diverse system is used to study the different processes of ice and hydrate crystallization and the mechanism by which the growth of these crystals is inhibited. The protocol describes how to prepare microfluidic devices, how to grow and control microscopic crystals in the microfluidic channels, and how the utilization of the flow of liquids around ice/hydrate crystals affords new insights into the crystallization of water.

The present protocol describes the crystallization of microscopic ice crystals and clathrate hydrates in microfluidic devices, enabling liquid exchange around the formed crystals. This provides unparalleled possibilities to examine the crystallization process and binding mechanisms of the inhibitors.

一种用于研究冰和包合物水合物结晶的微流控方法

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

生物学

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

基于AirID的植物蛋白质-蛋白质相互作用的邻近标记

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...