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不足以用于将来的实验或分析。蛋白质提取过程中快速工作，增加了可靠的蛋白酶抑制剂混合物，可以减少这样的蛋白损失。此外，重要的是，冷驯化期IBPS在实验室培养的最伟大的积累进行优化植物。 与IBPS工作时另一个常见的问题是，许多的测定的对温度和湿度敏感。在问候IRI分析中，在显微镜盖玻片湿气积聚可导致一层冰，使得冰晶体的可视化困难。使用除湿机和额外的干燥珠可以解决这个难题;然而，推荐的是，当实验室相对湿度水平是在70％以上该测定不进行。 在片分析，所呈现，是定性的。通过分析之前执行的稀释系列中，IRI端点可以建立，以确定在其中IBPS不再限制冰晶生长的浓度范围内。各种实验室还使用计算机软件来测量冰晶颗粒18的平均尺寸。如前面所指出，在测试IRI活动是一种有效的初始画面，确认冰结合活动应该通过确定TH的电平或通过直接可视化IBPS的吸附到冰晶16来建立。 IRI分析的一个显着的限制是若干非IBP分子已经鉴定了模拟IRI活性。这些分子可包括大分子蛋白质，酚醛苷，和合成聚合物，如聚乙烯醇19，20，21。其结果是，缓冲器控制应始终与样品运行，以确保任何污染物或添加剂不会导致所观察到的IRI活性。 而容易进行冰冷亲和纯化，当不正确地完成，其它蛋白质溶质和代谢物可占优势。一个重要的考虑是，冰晶生长率;通过降低冷却速度（ 即 ，-0.02℃/ h）时，冰半球生长更慢，任何非冰结合molecuLES是不太可能成为在冰晶格并入。污染分子的存在也可以通过确保只有50％的裂解物被结合到半球避免（ 即 50:50，液体至冰比）。如前所述，多轮冰亲和力还可以澄清冰部分并导致更高纯度的于样品中。值得注意的是，次级代谢产物通常在植物中生物和非生物胁迫条件下过量产生。在富含苯酚的植物组织的纯化中，如果黑褐色颜料持续，1.5％（V / V）的聚乙烯吡咯烷酮（PVP）和1.5％聚乙烯聚吡咯烷酮（W / V）（PVPP）可被加入到提取缓冲液，以中和次级代谢物。抗氧化添加剂如硫脲（5-10毫米）也可以使用19,21。 如果关于感兴趣IBP信息是已知的，另外的纯化步骤，如高压利镑色谱（HPLC）也可使用。修改以冰-亲和纯化，也可以考虑与已经报道以优化蛋白产量和更深入地了解IBP特性，包括更快的基于荧光冰壳纯化23和冰面亲和力（FIPA）24。 当优化，上述技术可以产生用于IBPS的鉴定纯化的蛋白质池;然而，其他高丰度蛋白质的非特异性结合可能是不可避免的。同时从早熟禾亚科家族共享序列同源性的25 IBPS，由于来自不同生物体之间IBPS高多样性，它通常是不可能通过序列同源性单独标识一个IBP。 IBPS的一个共同特征是已知结合冰，包括苏氨酸，丝氨酸和缬氨酸26个氨基酸的存在;识别肽序列富含第挖掘塞克斯·达塔时ESE残基可能是有用的。

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 border="0" cellpadding="0" cellspacing="0" width="1117">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL microcentrifugetubes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">05-408-129</td>
			<td style="width:631px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adjustable lab jack</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">S63080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop centrifuge</td>
			<td style="width:152px;">Desaga MC2</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brass probe</td>
			<td style="width:152px;">Custom built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera/ camera port</td>
			<td style="width:152px;">Canon</td>
			<td style="width:119px;"></td>
			<td>Canon Power Shot SX110 digitial camera; custom built microscope port</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cheesecloth</td>
			<td style="width:152px;">Purewipe/Fisher Scientific</td>
			<td style="width:119px;">06-665-25A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentration tubes (0.5 mL)</td>
			<td style="width:152px;">EMD Millipore</td>
			<td style="width:119px;">UFC501008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentration tubes (15 mL)</td>
			<td style="width:152px;">EMD Millipore</td>
			<td style="width:119px;">UFC900308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tubes (50 mL)</td>
			<td style="width:152px;">Thermo Fisher</td>
			<td style="width:119px;">AM12502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cooling block</td>
			<td style="width:152px;">VWR</td>
			<td style="width:119px;">13259</td>
			<td>Use a metal heating block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dehumidifier</td>
			<td style="width:152px;">Whirlpool</td>
			<td style="width:119px;"></td>
			<td>50 pint Energy Star dehumidifier; purchase from local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dessciation beads</td>
			<td style="width:152px;">t.h.e. Dessicant/VWR</td>
			<td style="width:119px;">EM-DX0017-2</td>
			<td>6-8 mesh size; 100% indicating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissection microscope</td>
			<td style="width:152px;">Olympus Tokyo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double walled glass bowl</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Purchase from local lab glassware supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dry ice</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Use local supplier; hazardous&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA-protease inhibitor tablets</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td>11836170001</td>
			<td>Roche cOmplete mini</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Green automotive ethylene glycol can be purchased from any local hardware store (i.e. Home Depot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexane</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">296090</td>
			<td>Anhydrous, 95%; hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Immersion oil</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">56822</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">JA10/20 centrifuge</td>
			<td style="width:152px;">Beckman</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large plastic petri dish</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid nitrogen</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Use local supplier; hazardous&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stir plate</td>
			<td style="width:152px;">Hanna Instruments</td>
			<td style="width:119px;">HI190M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope cover slides</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic tube</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Purchase PVC pipe from local hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polarized film</td>
			<td style="width:152px;">Edmund Optics</td>
			<td style="width:119px;">43-781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polystyrene foam</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Can be constructed from polystyrene shipping boxes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poreclain mortar and pestle</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">FB961</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVPP</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">77627</td>
			<td>110 &#181;m particle size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retort Stand</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">12-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small stir bar</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">14-513-51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Temperature-programmable water bath</td>
			<td style="width:152px;">VWR</td>
			<td style="width:119px;">13271-118</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vacuum grease</td>
			<td style="width:152px;">Dow Corning/Sigma Aldrich</td>
			<td style="width:119px;">Z273554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vinyl tubing</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></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年生黑麦草限制冰晶生长基本上IBPS转基因植物。 图4示出了使用经过两轮纯化表达IBPS多年生黑麦草提取物生长冰半球。在hemisphe第一轮纯化的高度后冰蚀刻重新表面表示IBPS已吸附到冰和改性的冰晶（ 图4A）的生长。在图4B所示的第二轮纯化具有更清晰的冰，表明存在污染分子的一部分除外。半球表面上冰 - 蚀刻指示IBPS仍然存在。 图5示出了SDS-PAGE以下冰亲和纯化，从多年生黑麦草收集的蛋白，并随后用银染色剂染色。许多蛋白质被鉴定具有七个不同的条带，对应于该吸附到冰中最丰富的蛋白质。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55302/55302fig1.jpg" /> 图1:图示装置16的示意图 。我们的设备<html> ED为冰晶（A）的单层的产生和对冰晶生长（B）的可视化。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55302/55302fig2.jpg" /> 图2:用于IBPS 16的冰-亲和纯化装置 。黄铜"冰手指"附接至可编程乙二醇浴被用于生长缓慢冰半球。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> g3.jpg" /&gt; 图3:使用IRI分析17中获得的代表性数据 。使用植物提取物上形成的冰晶体的单层在40倍放大倍率观察。从野生型拟南芥提取物不包含IBPS（-IBPs）进行比较，以表达来自黑麦草 （+ IBPS）IBPS转基因拟南芥的提取物。以下内容的18小时温育期在-4℃下，冰晶已经缓冲和野生型拟南芥样品中重结晶。与此相反，冰晶生长在表达IBP 拟南芥提取物中的限制。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55302/55302fig4.jpg" /><br/&gt; 图4:使用来自冷冻耐受草， 黑麦草收集IBP提取冰半球生长。后草提取物进行一轮冰亲和纯化的，冰半球的表面的冰蚀刻指示IBPS（A）的成功掺入。以除去额外的溶质，颜料和污染性蛋白质，使用了第二轮冰亲和力，导致更清晰的冰半球（B）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55302/55302fig5.jpg" /> 图5:从多年生黑麦草监测天然IBPS的纯化。以下两轮冰 - 亲和纯化和离心浓缩的，天然草提取物进行电泳在变性聚丙烯酰胺凝胶（SDS-PAGE），然后用银染可视化。泳道1:分子量蛋白质梯;泳道2:未稀释的提取物;泳道3-5:2,1:5，和1:10（样品:水）的比例在水凝胶上样用1之前稀释的样品。由于这个特定植物物种包含许多IBPS的，观察到多个频带，其可以对应于不同的同种型IBP。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

科研

生物化学

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

生理活性类囊体的分离及其在能量依赖性蛋白转运检测中的应用

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

用奇米蛋白构建功能蛋白区的鉴定

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...