Name: purification of human s100a12 and its ion-induced oligomers for immune cell stimulation
Uploaded: 2019-09-29T12:30:00
Description: Watch this Scientific Journal Video about Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation at JoVE.com

这项研究得到了蒙斯特大学医学院（KE121201至C.K.）和德国研究基金会（DFG，Fo354/3-1到D.F.）的校内创新医学研究项目的资助。

注：有关准备缓冲液和库存解决方案，请参阅补充表 1。
1.大肠杆菌中的蛋白质表达
<ol><li>
克隆
	<ol>
<li>将无标记的人类S100A12（NCBI参考序列：NP_005612.1）克隆成细菌表达载体pET11b。为了表达蛋白质，将结构转换为大肠杆菌BL21（DE3）。 </li>
	</ol>
</li>
	<li>
文化
	<ol>
<li>通过在 14 mL 圆底管中接种 1mL 生长培养基（LB ...

<ol>
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V., 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=Structure+of+the+human+S100A12-copper+complex:+implications+for+host-parasite+defence.">Structure of the human S100A12-copper complex: implications for host-parasite defence.</a> Acta Crystallographica Section D, Biological Crystallography. 59 (Pt 5), 859-867 (2003).</li><li>Moroz, O. V., Blagova, E. V., Wilkinson, A. J., Wilson, K. S., Bronstein, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structures+of+human+S100A12+in+apo+form+and+in+complex+with+zinc:+new+insights+into+S100A12+oligomerisation.">The crystal structures of human S100A12 in apo form and in complex with zinc: new insights into S100A12 oligomerisation.</a> Journal of Molecular Biology. 391 (3), 536-551 (2009).</li><li>Korndorfer, I. P., Brueckner, F., Skerra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+the+human+(S100A8/S100A9)2+heterotetramer,+calprotectin,+illustrates+how+conformational+changes+of+interacting+alpha-helices+can+determine+specific+association+of+two+EF-hand+proteins.">The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting alpha-helices can determine specific association of two EF-hand proteins.</a> Journal of Molecular Biology. 370 (5), 887-898 (2007).</li><li>Moroz, O. V., 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=Both+Ca2++and+Zn2++are+essential+for+S100A12+protein+oligomerization+and+function.">Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function.</a> BMC Biochemistry. 10, 11 (2009).</li><li>Baudier, J., Glasser, N., Gerard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ions+binding+to+S100+proteins.+I.+Calcium-+and+zinc-binding+properties+of+bovine+brain+S100+alpha+alpha,+S100a+(alpha+beta),+and+S100b+(beta+beta)+protein:+Zn2++regulates+Ca2++binding+on+S100b+protein.">Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein.</a> Journal of Biological Chemistry. 261 (18), 8192-8203 (1986).</li><li>Dell&#8217;Angelica, E. C., Schleicher, C. H., Santome, J. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAGE+mediates+a+novel+proinflammatory+axis:+a+central+cell+surface+receptor+for+S100/calgranulin+polypeptides.">RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides.</a> Cell. 97 (7), 889-901 (1999).</li><li>Foell, D., 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=Proinflammatory+S100A12+Can+Activate+Human+Monocytes+via+Toll-like+Receptor+4.">Proinflammatory S100A12 Can Activate Human Monocytes via Toll-like Receptor 4.</a> American Journal of Respiratory and Critical Care Medicine. 187 (12), 1324-1334 (2013).</li><li>Kessel, 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=Calcium+and+zinc+tune+autoinflammatory+Toll-like+receptor+4+signaling+by+S100A12.">Calcium and zinc tune autoinflammatory Toll-like receptor 4 signaling by S100A12.</a> Journal of Allergy and Clinical Immunology. 142 (4), 1370-1373 (2018).</li><li>Armaroli, G., 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=Monocyte-Derived+Interleukin-1beta+As+the+Driver+of+S100A12-Induced+Sterile+Inflammatory+Activation+of+Human+Coronary+Artery+Endothelial+Cells:+Implications+for+the+Pathogenesis+of+Kawasaki+Disease.">Monocyte-Derived Interleukin-1beta As the Driver of S100A12-Induced Sterile Inflammatory Activation of Human Coronary Artery Endothelial Cells: Implications for the Pathogenesis of Kawasaki Disease.</a> Arthritis &amp; Rheumatology. 71 (5), 792-804 (2019).</li><li>GE Healthcare. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Strategies for Protein Purification. Handbook. , (2010).</li><li>Jackson, E., Little, S., Franklin, D. S., Gaddy, J. A., Damo, S. M. <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+Antimicrobial+Activity+of+S100A12.">Expression, Purification, and Antimicrobial Activity of S100A12.</a> Journal of Visualized Experiments. (123), (2017).</li><li>Kiss, B., Ecsedi, P., Simon, M., Nyitray, L. <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+S100+Protein-Protein+Complexes.">Isolation and Characterization of S100 Protein-Protein Complexes.</a> Methods in Molecular Biology. 1929, 325-338 (2019).</li><li>Magalhaes, P. O., 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=Methods+of+endotoxin+removal+from+biological+preparations:+a+review.">Methods of endotoxin removal from biological preparations: a review.</a> Journal of Pharmacy and Pharmaceutical Sciences. 10 (3), 388-404 (2007).</li><li>Petsch, D., Anspach, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin+removal+from+protein+solutions.">Endotoxin removal from protein solutions.</a> Journal of Biotechnology. 76 (2-3), 97-119 (2000).</li><li>Heilmann, R. M., Suchodolski, J. S., Steiner, J. 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在此协议中，我们描述了人类S100A12的无标记细菌表达及其纯化以及分离成不同的由水因诱导的寡聚物，用于免疫细胞刺激。与发表论文的S100A12蛋白质纯化8，23，24，使用高CaCl2（25 mM）在疏水-相互作用色谱是对我们的知识的独特。应用浓度为 1 到 5 mM 的几种方案确实会产生纯蛋白质，但我们观察到，使用 25 mM CaCl2</...

S100A12是一种钙结合蛋白，主要由人类粒细胞产生。蛋白质在（全身）炎症期间过度表达，其血清水平，特别是在（自动）炎症疾病，如全身性青少年特发性关节炎（sJIA），家族地中海热（FMF）或川崎病（KD）可以通知疾病活动和对治疗的反应。根据模式识别受体 （PRRs），如收费受体 （TPR），先天免疫系统可以通过病原体相关分子模式 （PAMPs）（如脂多糖 （LPS） 或损害相关分子模式 （DamPs;也称为"报警器"）。DAM是内源性分子，如细胞蛋白、脂质或核酸1。DAMP 功能很好地描述了钙素蛋白家族 S100A8/A9 和 S100A122的成员，据报告，它们还作为二价金属-氢合抗菌肽3、4、 5，6.根据可用的抗气强度 S100A12 可以像 S100 系列的其他成员一样，排列成不同的同源体，直到最近，S100A12-寡头化对 PRR 相互作用（尤其是 TLR4）的影响还不得而知。
蛋白质的单体形式（92个氨基酸，10.2 kDa）由两个EF-手螺旋环螺旋结构组成，通过柔性连结器连接。C-终端 EF 手包含经典的 Ca2+绑定主题，而N端 EF 手则具有 S100 蛋白质特异性扩展环路结构（"伪 EF-手"），并揭示了减少的 Ca2+- 亲和力。S100A12 结合的 Ca2+可诱导蛋白质的 C-终点体发生重大构象变化，从而导致每个单体上接触疏水性斑块，并形成二分界面。因此，在生理条件下，由S100A12形成的最小四元结构是一种非共价二元结构（约21 kDa），其中单个单体处于反平行方向。当被安排为二聚体时，S100A12被报告给隔离Zn2+以及其他二价金属离子，例如，Cu2+具有高亲和力7。这些离子在S100A12二聚体界面由一个亚单位的氨基酸H15和D25以及抗平行的其他亚单元8、9、10的H89协调。虽然早期的研究表明，Zn2+负载 S100A12 可能诱导蛋白质的组织到同质四元体 （44 kDa） 和导致增加 Ca2+- 亲和力11，12，最近的金属滴定研究6建议Ca2+结合S100A12，以增加蛋白质对Zn2+的亲和力。一旦 S100A12 EF 指针被 Ca2+完全占用，则额外的 Ca2+被认为在二聚物之间结合，触发六项机形成（约 63 kDa）。六甲四聚体结构的结构与四合体结构明显不同。建议四联体接口被中断，产生新的二聚体-二聚体接口，有利于六项机形成10。S100A12几乎完全由人类粒细胞表达，约占所有细胞蛋白的5%。在其DAMP功能S100A12在历史上被描述为高级糖化最终产品（RAGE）的多配体受体的激动剂，然后称为细胞外新发现的RAGE结合蛋白（EN-RAGE）14。尽管我们早先报告生化S100A12对RAGE和TLR415结合，但我们最近证明人类单核细胞以TLR4依赖方式对S100A12刺激作出反应。这需要将S100A12排列到其Ca2+/Zn2+-诱导的六甲体结构16中。
在这里，我们描述了重组人类S100A12及其免疫细胞刺激的孔诱导寡聚物的纯化程序16，17。这是基于两步色谱策略，最初包括一个电一体交换柱，以分离和浓缩蛋白质，并去除散装污染（例如，内毒素/脂多糖）18。电一交换色谱树脂根据不同的净表面电荷分离蛋白质。对于酸性蛋白质，如S100A12（等电点5.81），pH值为8.5的缓冲系统和强的黄极交换树脂可产生良好的分离。结合蛋白用高盐缓冲梯度洗脱。随着洗脱缓冲液中离子强度负离子的增加，与树脂表面的蛋白质竞争。蛋白质根据其净电荷单独洗出，因此，本文描述的缓冲液允许分离和浓缩过度表达的S100A12蛋白。由于脂多糖中的带负电荷，这些分子还与阴极交换树脂结合。然而，其较高的净电荷导致在应用高盐梯度的后期洗脱。为抛光目的介绍了纯化程序的第二步。这利用S100A12的钙结合能力，并去除疏水相互作用柱上剩余的杂质。S100A12 的钙结合会导致形成变化和在蛋白质表面接触疏水性斑块。在这种情况下，S100A12 与树脂的疏水表面相互作用。当EDTA的钙合合后，这种相互作用被逆转。在离子（尤其是钙和锌）存在的情况下，S100A12 可排列成同质寡聚物。为了研究不同寡聚物的结构-功能关系，我们用化学交联器稳定了二聚体、四甲和六甲重孔S100A12，并在尺寸排除色谱柱上分离了复合物。最后，对纯化蛋白及其电子诱导寡聚物的功能和生物活性进行了分析，对S100A12和LPS刺激的单核细胞的细胞因子释放进行了比较。
到目前为止，已经描述了各种净化S100A12的方法。例如，Jackson等人19日通过一个电子交换柱和随后的尺寸排除色谱，发表了一种纯化协议。在大小排除列上纯化抛光可产生良好的效果，但由于加载量有限等原因，可伸缩性不够灵活。由Kiss等人20号发表的另一种方法，将通过Ni2+亲和柱纯化标记蛋白描述为第一步，然后是酶裂解，以去除标记和进一步纯化步骤。与上述研究19、20相反，本协议中描述的所产生的蛋白质被确定为免疫细胞实验。因此，细菌培养的残留内毒素污染是一个挑战。虽然到目前为止已经描述了不同的内毒素去除方法，但没有统一的方法对任何给定的蛋白质溶液21，22同样有效。
总之，我们的协议将细菌系统中无标签表达的优点与高效的内毒素去除和纯蛋白的高产量相结合。

<table>
	<tbody>
		<tr>
			<td>pET11b vector</td>
			<td>Novagen</td>
			<td>&#160;</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3) competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>&#160;</td>
			<td>&#160;</td>
			<td>&#160;</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>100 x Non-essential amino acids</td>
			<td>Merck</td>
			<td>K 0293</td>
			<td></td>
		</tr>
		<tr>
			<td>25% HCl</td>
			<td>Carl Roth</td>
			<td>X897.1</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8722;20% Mini-PROTEAN TGX Protein Gels</td>
			<td>BioRad</td>
			<td>4561093</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Carl Roth</td>
			<td>HP62.1</td>
			<td></td>
		</tr>
		<tr>
			<td>BS3 (bis(sulfosuccinimidyl)suberate) - 50 mg</td>
			<td>ThermoFisher Scientific</td>
			<td>21580</td>
			<td></td>
		</tr>
		<tr>
			<td>Calciumchlorid Dihydrat</td>
			<td>Carl Roth</td>
			<td>5239.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Briliant Blue R250 Destaining Solution</td>
			<td>BioRad</td>
			<td>1610438</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Briliant Blue R250 Staining Solution</td>
			<td>BioRad</td>
			<td>1610436</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human Monocyte Enrichment Kit</td>
			<td>Stemcell</td>
			<td>19059</td>
			<td>Magnetic negative cell isolation kit</td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate</td>
			<td>Carl Roth</td>
			<td>8043.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.3</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoLISA</td>
			<td>Hyglos</td>
			<td>609033</td>
			<td>Endotoxin detection assay</td>
		</tr>
		<tr>
			<td>Endotoxin-Free Ultra Pure Water</td>
			<td>Sigma-Aldrich</td>
			<td>TMS-011-A</td>
			<td>Ultrapure water for preparation of endotoxin-free buffers</td>
		</tr>
		<tr>
			<td>EndoTrap red</td>
			<td>Hyglos</td>
			<td>321063</td>
			<td>Endotoxin removal resin</td>
		</tr>
		<tr>
			<td>FBS (heat-inactivated)</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium</td>
			<td>ThermoFisher Scientific</td>
			<td>14175053</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Carl Roth</td>
			<td>9105</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes (high quality, endotoxin testet)</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>hTNF-alpha - OptEia ELISA Set</td>
			<td>BD</td>
			<td>555212</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG (isopropyl-&#223;-D-thiogalactopyranosid)</td>
			<td>Carl Roth</td>
			<td>CN08.1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Merck</td>
			<td>K 0282</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Medium</td>
			<td>Carl Roth</td>
			<td>X968.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from E. coli O55:B5</td>
			<td>Merck</td>
			<td>L6529</td>
			<td></td>
		</tr>
		<tr>
			<td>Pancoll, human</td>
			<td>PAN Biotech</td>
			<td>P04-60500</td>
			<td>Separation solution (density gradient centrifugation)</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (10.000 U/ml)</td>
			<td>Merck</td>
			<td>A 2212</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenyl Sepharose High Performance</td>
			<td>GE Healthcare</td>
			<td>17-1082-01</td>
			<td>Resin for hydrophobic interaction chromatography</td>
		</tr>
		<tr>
			<td>Polymyxin B</td>
			<td>Invivogen</td>
			<td>tlrl-pmb</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor tablets</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Sepharose Fast Flow</td>
			<td>GE Healthcare</td>
			<td>17-0510-01</td>
			<td>Resin for anion-exchange chromatography</td>
		</tr>
		<tr>
			<td>RoboSep buffer</td>
			<td>Stemcell</td>
			<td>20104</td>
			<td>Cell separation buffer (section 5.1.4)</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Merck</td>
			<td>F 1215</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Carl Roth</td>
			<td>3957.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Carl Roth</td>
			<td>P031.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Carl Roth</td>
			<td>4855.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc chloride</td>
			<td>Carl Roth</td>
			<td>T887</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0,45 &#181;m syringe filter</td>
			<td>Merck</td>
			<td>SLHA033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL roundbottom tubes</td>
			<td>BD</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>2 L Erlenmyer flask</td>
			<td>Carl Roth</td>
			<td>LY98.1</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well suspension plates</td>
			<td>Greiner</td>
			<td>662102</td>
			<td></td>
		</tr>
		<tr>
			<td>5 L measuring beaker</td>
			<td>Carl Roth</td>
			<td>CKN3.1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL high-speed centrifuge tubes</td>
			<td>Eppendorf</td>
			<td>3,01,22,178</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Unit MWCO 3 kDa</td>
			<td>Merck</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Unit MWCO 50 kDa</td>
			<td>Merck</td>
			<td>UFC905024</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish (100 mm)</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis Tubing Closures</td>
			<td>Spectrum</td>
			<td>132738</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep magnet &#39;The Big Easy`</td>
			<td>Stemcell</td>
			<td>18001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction collector tubes 5 mL</td>
			<td>Greiner</td>
			<td>115101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lumox film, 25 &#181;m, 305 mm x 40 m</td>
			<td>Sarstedt</td>
			<td>94,60,77,316</td>
			<td>Film for monocyte culture plates</td>
		</tr>
		<tr>
			<td>Spectra/Por Dialysis Membrane (3.5 kDa)</td>
			<td>Spectrum</td>
			<td>132724</td>
			<td></td>
		</tr>
		<tr>
			<td>Steritop filter unit</td>
			<td>Merck</td>
			<td>SCGPT01RE</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C Incubator (with shaking)</td>
			<td>New Brunswick Scientific</td>
			<td>Innova 42</td>
			<td></td>
		</tr>
		<tr>
			<td>&#196;KTA purifier UPC 10</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>FPLC System</td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>GE Healthcare</td>
			<td>Frac-920</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (with rotor A-4-81)</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed angle rotor</td>
			<td>Eppendorf</td>
			<td>F-34-6-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Protean Tetra Cell</td>
			<td>BioRad</td>
			<td>1658000EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoPhotometer</td>
			<td>Implen</td>
			<td>P330</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Brandelin</td>
			<td>UW2070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence reader</td>
			<td>Tecan</td>
			<td>infinite M200PRO</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Knick</td>
			<td>765</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of human s100a12 and its ion-induced oligomers for immune cell stimulation

在此协议中，我们描述了一种从大肠杆菌培养中纯化人体钙结合蛋白S100A12及其由水因诱导的寡聚物以进行免疫细胞刺激的方法。该协议基于两步色谱策略，其中包括在一个海龙交换色谱柱上的蛋白质预纯化，以及随后对疏水性相互作用柱的抛光步骤。该策略以可管理的成本生产高纯度和产量的 S100A12 蛋白质。对于免疫细胞的功能检测，最终残留内毒素污染需要仔细监测和进一步清洁步骤，以获得无内毒素蛋白质。大多数内毒素污染可以通过电一孔交换色谱法排除。为了消耗残留污染，此协议描述了使用离心过滤器的去除步骤。根据可用的抗子强体 S100A12 可以排列成不同的同源体。为了研究结构和功能之间的关系，本方案进一步描述了S100A12蛋白的电处理，然后化学交联以稳定S100A12寡聚物及其随后通过尺寸排除分离相 色 谱 法。最后，我们描述了一种基于细胞的测定，它证实了纯化蛋白的生物活性，并证实了LPS的无制备。

在AIEX列（图1A-C）和随后的钙依赖性HIC（图2A，B）上预纯后，获得高纯蛋白（图2C）。此外，内毒素的测量显示LPS去除成功。AIEX 之后的 LPS 含量在高于测定检测限值（即高于 500 EU/mL）的 1：10 稀释度下测量。通过 50 kDa 过滤单元进行第一次过滤后，LPS 含量降至 1 欧/mL。在使用 3 kDa 滤光片和通过 50 kDa 进行?...

Watch this Scientific Journal Video about Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation at JoVE.com

Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation

该协议描述了一种用于重组无标记标记的钙结合蛋白S100A12及其用于人类单核细胞刺激测定的由水因诱导的寡聚物的纯化方法。

人类S100A12及其用于免疫细胞刺激的Ion诱导寡聚物的纯化

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for ...

该协议描述了人类钙结合蛋白S100A12的完整纯化过程，特别是对于下游研究的影响，它具有不同的离子诱导的寡聚物。通过这项技术，我们能够保持高产量的非常纯净和无内毒素的蛋白质。大小排除色谱柱上的化学交链接和分离是生成同寡聚蛋白进行进一步功能测定的令人信服的方法。我们的技术使我们能够将S100A12作为模式识别受体配体和艺术信息机械化，以理想地开发新的针对性干预措施并改进诊断分析。除了观看此视频之外，我们建议仔细阅读协议，因为我们描述了一个复杂的多步骤过程。首先，将透析管切成适当的长度，大约30厘米，并额外的空间供空气使用。将膜浸泡在去电化水中 15 至 30 分钟，以去除甘油。为了降低清除蛋白溶液的粘度，用25毫升AIEX缓冲液 A.将第一个闭合附到油管上稀释溶液。将样品装入膜中，将第二个封口从管的顶端连接至少一厘米。在四摄氏度的冷却室里，将装有AIEX缓冲液的容器放在搅拌盘上，加入搅拌棒，并填充充满蛋白质溶液的膜。调整旋转样品的速度，以便旋转搅拌杆没有干扰。在 4 摄氏度下，Dialyze 持续 12 至 24 小时，然后用免费的预冷缓冲器 A 更换拨号缓冲器，并至少再持续 4 小时。通过 0.45 微米过滤装置将透析蛋白溶液从管中转移到 50 毫升管中。使用常规维护启动 FPLC。将列缓冲器 AIEX A 和 AIEX B 连接到包含柱的阀和 Anion 交换树脂到 FPLC 的柱端口。相应地调整一般色谱参数。在FPLC软件中启动AIEX程序，然后选择缓冲液 A 的均衡体积。在洗脱过程中收集两毫升分数。完成运行后分析洗小分数。为此，在 Coomassie 染色 15% SDS 页面上加载每个分数的 10 微升，并识别包含 S100A12 的分数。将这些 S100A12 中含透析分数的池到 TRIS 缓冲盐水中。为了继续蛋白质纯化，在蛋白质透析后，在样品中加入氯化钙，最终浓度为25毫摩尔，这将有助于下一步将S100A12与树脂结合。然后，通过倾水尺寸为 0.45 微米的过滤器过滤样品。将 HIC 缓冲液和样品平衡到 4 摄氏度。接下来，将列缓冲区 HIC A 和 B 以及列连接到系统并调整参数。开始方法，使用一到两列缓冲区 HIC A 的列进行平衡，并加载样本，并且具有 UV 信号的洗涤未绑定样本块再次达到基线级别。然后开始使用含有缓冲 EDTA 的钙溷合器进行洗脱。在洗脱过程中收集两毫升的峰值分数。在分析 Coomassie 染色 15% SDS 页面上每个分数的 10 微升后，识别纯 S100A12 分数。将这些分数与 HBS 池起并拨号。首先，将 15 毫升样品加载到 50 千吨离心过滤器单元上。在10摄氏度下以3200倍g离心约10分钟。将过滤过的流经转移到冰上的新鲜容器中。现在，使用一毫升的HBS冲洗滤膜，并稀释过滤片尖中的浓缩溶液。再次离心，在流经中尽可能多地恢复蛋白质。要过滤溶液，请根据需要重复样品加载和离心。接下来，将含有流经的S100A12浓缩，装入三千吨离心过滤器，以3200倍g、10摄氏度的离心机约30分钟。现在，音量减少到初始加载体积的 1/5 至 1/10。将浓缩溶液转移到新管中。根据需要经常重复浓度步骤。化学交链接后，根据手稿，进行大小排除色谱。平衡 HBS 中的列，加载样品，并在运行过程中收集 1 到 2 毫升的峰值分数。在 4 到 20% 梯度 SDS 页上分析这些分数后，将分数与所需蛋白质复合物的主要波段进行池化。然后像以前一样集中解决方案。通过密度梯度离心将单核细胞与人毛涂层分离，首先将分离溶液与室温平衡。将 20 毫升转移到 15 毫升离心管中，每层两根管。用HSS稀释人体布衣中的血液，在分离介质上小心地将这种混合物的体积稀释到60毫升和30毫升。在室温下以 550 倍 g 离心 35 分钟。关闭离心机的制动器。离心后，单核外周血细胞直接位于分离介质顶部。使用移液器，将这些细胞转移到一个50毫升的新鲜离心管中。用HSSS填充管至50毫升，以170倍g离心10分钟。通过移液，吸进上一提液，并在少量HSSS中重新暂停细胞颗粒。然后，将管充满50毫升，以290倍g离心10分钟。再次吸上一月。将细胞在50毫升的HSS中重新暂停，以170倍g的离心机，10分钟。吸升液，将细胞颗粒重新在一毫升分离缓冲液的小体积中，计算细胞数，并将缓冲液添加到每毫升七个细胞的浓度的五倍十分之一。对于从单核外周血细胞分离的单核细胞，请使用磁性负细胞分离试剂盒，并遵循制造商的协议。使用自动细胞计数器计算单核细胞，并在单核介质中重新暂停，浓度为每毫升六个细胞的浓度为 2 倍 10。培养单核细胞，布置一个细胞培养皿与疏水气体渗透膜适合悬浮细胞。将板在紫外线下放置约 30 分钟进行灭菌。将15至25毫升的细胞悬浮液转移到这些培养板，让它们在37摄氏度和5%的二氧化碳下过夜。在AIEX柱上预纯后，随后获得高纯蛋白。作为一种额外的控制，人类单核细胞受到LPS的刺激，并产生野生型蛋白质。蛋白质暴露在不同的离子中会导致不同S100A12寡聚物的排列。增加锌浓度诱导S100A12排列成三角和六角板分离在四至20%库马西染色SDS页面。当在交链接之前应用过量的离子时，它导致寡聚体平衡的显著转移。S100A12 在存在 25 毫摩尔钙或 25 毫摩尔钙和 1 毫摩尔锌的情况下是交叉链接的。在HS缓冲液中与25毫摩尔氯化钙和1毫拉氯化锌进行交对后，将六角体和四分之一分液分离。利四甲和二丁二甲在HS缓冲液中与25毫摩尔氯化钙分离。在 Coomassie 染色的 4 到 15% 梯度 SDS 页上分离后，池和浓缩寡聚物的一个示例显示了二聚体、四边板和六角体。六核S100A12的单细胞刺激导致明显的TNFα释放。去除内毒素后，在随后生成和纯化寡聚物时，不要将污染重新引入样品中。可想而知，任何与S100A12寡聚化相关的过程的下游分析。同样，可以分析影响寡聚化过程本身的氨基酸和离子条件。我们目前使用S100A12寡聚物根据所述协议分离，以开发特定的肽配体和抗体，以阻止蛋白质通过TLF4的信号，并开发和改进诊断分析。

Research

JoVE Journal

Immunology and Infection

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

科研

免疫与感染

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...