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
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?...

<ol>
	<li>Sidebottom, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-stable+antifreeze+protein+from+grass.">Heat-stable antifreeze protein from grass.</a> Nature. 406 (6793), 256 (2000).</li><li>Duman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+and+ice+nucleator+proteins+in+terrestrial+arthropods.">Antifreeze and ice nucleator proteins in terrestrial arthropods.</a> Annu Rev Physiol. 63, 327-357 (2001).</li><li>Davies, P. L., Hew, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+fish+antifreeze+proteins.">Biochemistry of fish antifreeze proteins.</a> FASEB J. 4 (8), 2460-2468 (1990).</li><li>Gilbert, J. A., Hill, P. J., Dodd, C. E., Laybourn-Parry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+antifreeze+protein+activity+in+Antarctic+lake+bacteria.">Demonstration of antifreeze protein activity in Antarctic lake bacteria.</a> Microbiology. 150 (Pt 1), 171-180 (2004).</li><li>Knight, C. A., Cheng, C. C., DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+alpha-helical+antifreeze+peptides+on+specific+ice+crystal+surface+planes.">Adsorption of alpha-helical antifreeze peptides on specific ice crystal surface planes.</a> Biophys. L. 59 (20), 409-418 (1991).</li><li>Yeh, Y., Feeney, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+proteins:+structures+and+mechanisms+of+function.">Antifreeze proteins: structures and mechanisms of function.</a> Chem. Rev. 96 (2), 601-618 (1996).</li><li>Smolin, N., Daggett, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+ice-like+water+structure+on+the+surface+of+an+antifreeze+protein.">Formation of ice-like water structure on the surface of an antifreeze protein.</a> J. Phys. Chem. B. 112 (19), 6193-6202 (2008).</li><li>Graham, L. A., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycine-rich+antifreeze+proteins+from+snow+fleas.">Glycine-rich antifreeze proteins from snow fleas.</a> Science. 310 (5747), 461 (2005).</li><li>Hawes, T. C., Marshall, C. J., Wharton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+proteins+in+the+Antarctic+springtail,+Gressittacantha+terranova.">Antifreeze proteins in the Antarctic springtail, Gressittacantha terranova.</a> J. Comp. Physiol. B. 181 (6), 713-719 (2011).</li><li>Basu, K., Graham, L. A., Campbell, R. L., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flies+expand+the+repertoire+of+protein+structures+that+bind+ice.">Flies expand the repertoire of protein structures that bind ice.</a> Proc. Natl. Acad. Sci. U.S.A. 112 (3), 737-742 (2015).</li><li>Guo, S., Garnham, C. P., Whitney, J. C., Graham, L. A., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluation+of+a+Bacterial+Antifreeze+Protein+as+an+Adhesin+with+Ice-Binding+Activity.">Re-evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity.</a> PLoS One. 7 (11), e48805 (2012).</li><li>Marshall, C. B., Fletcher, G. L., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactive+antifreeze+protein+in+a+fish.">Hyperactive antifreeze protein in a fish.</a> Nature. 429 (6988), 153 (2004).</li><li>Zhang, D. Q., Liu, B., Feng, D. R., He, Y. M., Wang, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+purification+and+antifreeze+activity+of+carrot+antifreeze+protein+and+its+mutants.">Expression, purification and antifreeze activity of carrot antifreeze protein and its mutants.</a> Protein Expr. Purif. 35 (2), 257-263 (2007).</li><li>Gupta, R., Dreswal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+temperature+stress+modulated+secretome+analysis+and+purification+of+antifreeze+protein+from+Hippophae+rhamnoides,+a+Himalayan+wonder+plant.">Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant.</a> Proteome Res. 11 (5), 2684-2696 (2012).</li><li>Kuiper, M. J., Lankin, C., Gauthier, S. Y., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+antifreeze+proteins+by+adsorption+to+ice.">Purification of antifreeze proteins by adsorption to ice.</a> Biochem. Biphys. Res. Commun. 300 (3), 645-648 (2003).</li><li>Middleton, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+ice-binding+proteins+from+higher+plants.">Isolation and characterization of ice-binding proteins from higher plants.</a> Methods Mol. Biol. 1166, 255-277 (2014).</li><li>Bredow, M., Vanderbeld, B., 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=Ice-binding+proteins+confer+freezing+tolerance+in+transgenic+Arabidopsis+thaliana.">Ice-binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana.</a> Plant Biotechnol. J. , (2016).</li><li>Olijve, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+rapid+ice+crystal+growth+through+nonbasal+plane+adsorption+of+antifreeze+proteins.">Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 113 (14), 3740-3745 (2016).</li><li>Zakharov, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+Recrystallization+in+a+Solution+of+a+Cryoprotector+and+Its+inhibition+by+a+Protein:+Synchrotron+X-Ray+Diffraction+Study.">Ice Recrystallization in a Solution of a Cryoprotector and Its inhibition by a Protein: Synchrotron X-Ray Diffraction Study.</a> J. Pharm. Sci. 105 (7), 2129-2138 (2016).</li><li>Capicciotti, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+ice+recrystallization+inhibitors+enable+freezing+of+human+red+blood+cells+with+reduced+glycerol+concentrations.">Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations.</a> Sci. Rep. 5, 9692 (2015).</li><li>Knight, C. A., Cheng, C. C., DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+alpha-helical+antifreeze+peptides+on+specific+ice+crystal+surface+planes.">Adsorption of alpha-helical antifreeze peptides on specific ice crystal surface planes.</a> Biophys. J. 59 (2), 409-418 (1991).</li><li>Van Driessche, E., Beeckmans, S., Dejaegere, R., Kanarek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiourea:+the+antioxidant+of+choice+for+the+purification+of+proteins+from+phenol+rich+plant+tissues.">Thiourea: the antioxidant of choice for the purification of proteins from phenol rich plant tissues.</a> Anal. Biochem. 141 (1), 184-188 (1984).</li><li>Marshall, C. J., Basu, K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-shell+purification+of+ice-binding+proteins.">Ice-shell purification of ice-binding proteins.</a> Cryobiology. 72 (3), 258-263 (2016).</li><li>Basu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+ice-binding+planes+of+antifreeze+proteins+by+fluorescence-based+ice+plane+affinity.">Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.</a> J. Vis. Exp. (83), e51185 (2014).</li><li>Sandve, S. R., Rudi, H., Asp, T., Rognli, O. 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>

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 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?...

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 ...

Research

JoVE Journal

Biochemistry

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

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

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

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 ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

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 ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

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

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

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

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

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

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

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 ...