Name: quantification of metal leaching in immobilized metal affinity chromatography
Uploaded: 2020-01-17T16:00:00
Description: Watch this Scientific Journal Video about Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography at JoVE.com

本材料基于国家科学基金会在格兰特MCB-1817448下支持的工作，以及托马斯·F和凯特·米勒·杰弗里斯纪念信托基金、美国银行、受托人和指定捐赠者黑兹尔·索普·卡曼和乔治·盖伊·卡曼的奖项。信任。

1. 测试组件制备
<ol><li>确定使用 280 nm 的光学吸收度或蛋白质定量的替代方法测定要测定的色谱分数，以确定蛋白质富集度分数。 注：为了这项工作，我们使用了二极管阵列UV-Vis分光光度计。为了增加吞吐量，可以使用能够测量 UV-Vis 吸光度的板式读取器。</li>
	<li>制备必要的测定组件<ol>
<li>在 pH 值介于 7 和 12 之间准备或获取 10-100 mM 缓冲液（"样品缓冲器"）。 注：在中性或基本pHH下，常见...

<ol>
	<li>Porath, J., Carlsson, J. A. N., Olsson, I., Belfrage, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+chelate+affinity+chromatography,+a+new+approach+to+protein+fractionation.">Metal chelate affinity chromatography, a new approach to protein fractionation.</a> Nature. 258 (5536), 598-599 (1975).</li><li>Block, H., 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=Immobilized-Metal+Affinity+Chromatography+(IMAC):+A+Review.">Immobilized-Metal Affinity Chromatography (IMAC): A Review.</a> Methods in Enzymology. 463, 439-473 (2009).</li><li>Takeda, N., Matsuoka, T., Gotoh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiality+of+IMAC+as+sample+pretreatment+tool+in+food+analysis+for+veterinary+drugs.">Potentiality of IMAC as sample pretreatment tool in food analysis for veterinary drugs.</a> Chromatographia. 72 (1/2), 127-131 (2010).</li><li>Felix, 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=Identification+of+serum+proteins+involved+in+pancreatic+cancer+cachexia.">Identification of serum proteins involved in pancreatic cancer cachexia.</a> Life sciences. 88 (5-6), 218-225 (2011).</li><li>Wu, 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=Surface+enhanced+laser+desorption/ionization+profiling:+New+diagnostic+method+of+HBV-related+hepatocellular+carcinoma.">Surface enhanced laser desorption/ionization profiling: New diagnostic method of HBV-related hepatocellular carcinoma.</a> Journal of Gastroenterology and Hepatology. 24 (1), 55-62 (2009).</li><li>Goldsmith, J. O., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+bacterial+photosynthetic+reaction+centers+with+an+engineered+poly-histidine+tag.">Rapid isolation of bacterial photosynthetic reaction centers with an engineered poly-histidine tag.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1276 (3), 171-175 (1996).</li><li>Guergova-Kuras, M., 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=Expression+and+one-step+purification+of+a+fully+active+polyhistidine-tagged+cytochrome+bc1+complex+from+Rhodobacter+sphaeroides.">Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides.</a> Protein Expression and Purification. 15 (3), 370-380 (1999).</li><li>Mitchell, D. M., Gennis, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+wildtype+and+mutant+cytochrome+c+oxidase+from+Rhodobacter+sphaeroides+by+Ni(2+)-NTA+affinity+chromatography.">Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni(2+)-NTA affinity chromatography.</a> FEBS Letters. 368 (1), 148-150 (1995).</li><li>Tian, H., White, S., Yu, L., Yu, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+head+domain+movement+of+the+rieske+iron-sulfur+protein+in+electron+transfer+reaction+of+the+cytochrome+bc1+complex.">Evidence for the head domain movement of the rieske iron-sulfur protein in electron transfer reaction of the cytochrome bc1 complex.</a> Journal of Biological Chemistry. 274 (11), 7146-7152 (1999).</li><li>Tian, H., Yu, L., Mather, M. W., Yu, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexibility+of+the+neck+region+of+the+rieske+iron-sulfur+protein+is+functionally+important+in+the+cytochrome+bc1+complex.">Flexibility of the neck region of the rieske iron-sulfur protein is functionally important in the cytochrome bc1 complex.</a> Journal of Biological Chemistry. 273 (43), 27953-27959 (1998).</li><li>Louie, A. Y., Meade, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+complexes+as+enzyme+inhibitors.">Metal complexes as enzyme inhibitors.</a> Chemical Reviews. 99 (9), 2711-2734 (1999).</li><li>Tam&#225;s, M. J., Sharma, S. K., Ibstedt, S., Jacobson, T., Christen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heavy+Metals+and+Metalloids+As+a+Cause+for+Protein+Misfolding+and+Aggregation.">Heavy Metals and Metalloids As a Cause for Protein Misfolding and Aggregation.</a> Biomolecules. 4 (1), 252-267 (2014).</li><li>Gerencser, L., Maroti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retardation+of+proton+transfer+caused+by+binding+of+the+transition+metal+ion+to+the+bacterial+reaction+center+is+due+to+pKa+shifts+of+key+protonatable+residues.">Retardation of proton transfer caused by binding of the transition metal ion to the bacterial reaction center is due to pKa shifts of key protonatable residues.</a> Biochemistry. 40 (6), 1850-1860 (2001).</li><li>Klishin, S. S., Junge, W., Mulkidjanian, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash-induced+turnover+of+the+cytochrome+bc1+complex+in+chromatophores+of+Rhodobacter+capsulatus:+binding+of+Zn2++decelerates+likewise+the+oxidation+of+cytochrome+b,+the+reduction+of+cytochrome+c1+and+the+voltage+generation.">Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn2+ decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1553 (3), 177-182 (2002).</li><li>Link, T. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+ions+inhibit+the+QP+center+of+bovine+heart+mitochondrial+bc1+complex+by+blocking+a+protonatable+group.">Zinc ions inhibit the QP center of bovine heart mitochondrial bc1 complex by blocking a protonatable group.</a> Journal of Biological Chemistry. 270 (42), 25001-25006 (1995).</li><li>Block, H., Kubicek, J., Labahn, J., Roth, U., Sch&#228;fer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+comprehensive+quality+control+of+recombinant+human+Interleukin-1beta:+a+case+study+for+a+process+development+strategy.">Production and comprehensive quality control of recombinant human Interleukin-1beta: a case study for a process development strategy.</a> Protein Expression and Purification. 57 (2), 244-254 (2008).</li><li>Kokhan, O., Shinkarev, V. P., Wraight, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+imidazole+to+the+heme+of+cytochrome+c1+and+inhibition+of+the+bc1+complex+from+Rhodobacter+sphaeroides:+II.+Kinetics+and+mechanism+of+binding.">Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: II. Kinetics and mechanism of binding.</a> Journal of Biological Chemistry. 285 (29), 22522-22531 (2010).</li><li>Kokhan, O., Shinkarev, V. P., Wraight, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+imidazole+to+the+heme+of+cytochrome+c1+and+inhibition+of+the+bc1+complex+from+Rhodobacter+sphaeroides:+I.+Equilibrium+and+modeling+studies.">Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: I. Equilibrium and modeling studies.</a> Journal of Biological Chemistry. 285 (29), 22513-22521 (2010).</li><li>Bornhorst, J. A., Falke, J. J. <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+proteins+using+polyhistidine+affinity+tags.">Purification of proteins using polyhistidine affinity tags.</a> Methods in Enzymology. 326, 245-254 (2000).</li><li>Kokhan, O., Marzolf, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+transition+metal+leaching+in+metal+affinity+chromatography+with+hydroxynaphthol+blue.">Detection and quantification of transition metal leaching in metal affinity chromatography with hydroxynaphthol blue.</a> Analytical Biochemistry. 582, 113347 (2019).</li><li>Doyle, C., Naser, D., Bauman, H., Rumfeldt, J., Meiering, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+method+for+simultaneous+measurement+of+zinc+and+copper+in+metalloproteins+using+4-(2-pyridylazo)resorcinol.">Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol.</a> Analytical Biochemistry. 579, 44-56 (2019).</li><li>Furrer, J., Smith, G. S., Therrien, B., Gasser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inorganic Chemical Biology. , (2014).</li><li>Hogeling, S. M., Cox, M. T., Bradshaw, R. M., Smith, D. P., Duckett, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+proteins+in+whole+blood,+plasma+and+DBS,+with+element-labelled+antibody+detection+by+ICP-MS.">Quantification of proteins in whole blood, plasma and DBS, with element-labelled antibody detection by ICP-MS.</a> Analytical Biochemistry. 575, 10-16 (2019).</li><li>Yamasaki, S., Tsumura, A., Takaku, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultratrace+Elements+in+Terrestrial+Water+as+Determined+by+High-Resolution+ICP-MS.">Ultratrace Elements in Terrestrial Water as Determined by High-Resolution ICP-MS.</a> Microchemical Journal. 49 (2), 305-318 (1994).</li><li>Brittain, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+Formation+Between+Hydroxy+Naphthol+Blue+and+First+Row+Transition+Metal+Cyanide+Complexes.">Complex Formation Between Hydroxy Naphthol Blue and First Row Transition Metal Cyanide Complexes.</a> Analytical Letters. 10 (13), 1105-1113 (1977).</li><li>Brittain, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+Transition+Metal+Ions+by+the+Calcium+Indicator+Hydroxy+Naphthol+Blue.">Binding of Transition Metal Ions by the Calcium Indicator Hydroxy Naphthol Blue.</a> Analytical Letters. 11 (4), 355-362 (1978).</li><li>Temel, N. K., Sertakan, K., G&#252;rkan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconcentration+and+Determination+of+Trace+Nickel+and+Cobalt+in+Milk-Based+Samples+by+Ultrasound-Assisted+Cloud+Point+Extraction+Coupled+with+Flame+Atomic+Absorption+Spectrometry.">Preconcentration and Determination of Trace Nickel and Cobalt in Milk-Based Samples by Ultrasound-Assisted Cloud Point Extraction Coupled with Flame Atomic Absorption Spectrometry.</a> Biological Trace Element Research. 186 (2), 597-607 (2018).</li><li>Ferreira, S. L. C., Santos, B. F., de Andrade, J. B., Costa, A. C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+and+derivative+spectrophotometric+determination+of+nickel+with+hydroxynaphthol+blue.">Spectrophotometric and derivative spectrophotometric determination of nickel with hydroxynaphthol blue.</a> Microchimica Acta. 122 (1), 109-115 (1996).</li><li>Denisov, I. G., Grinkova, Y. V., Lazarides, A. A., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+Self-Assembly+of+Monodisperse+Phospholipid+Bilayer+Nanodiscs+with+Controlled+Size.">Directed Self-Assembly of Monodisperse Phospholipid Bilayer Nanodiscs with Controlled Size.</a> Journal of the American Chemical Society. 126 (11), 3477-3487 (2004).</li><li>Grinkova, Y. V., Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+extended+membrane+scaffold+proteins+for+self-assembly+of+soluble+nanoscale+lipid+bilayers.">Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers.</a> Protein Engineering, Design and Selection. 23 (11), 843-848 (2010).</li><li>Bonta, M., Hegedus, B., Limbeck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+dried-droplets+deposited+on+pre-cut+filter+paper+disks+for+quantitative+LA-ICP-MS+imaging+of+biologically+relevant+minor+and+trace+elements+in+tissue+samples.">Application of dried-droplets deposited on pre-cut filter paper disks for quantitative LA-ICP-MS imaging of biologically relevant minor and trace elements in tissue samples.</a> Analytica Chimica Acta. 908, 54-62 (2016).</li><li>Olmedo, P., 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=Validation+of+a+method+to+quantify+chromium,+cadmium,+manganese,+nickel+and+lead+in+human+whole+blood,+urine,+saliva+and+hair+samples+by+electrothermal+atomic+absorption+spectrometry.">Validation of a method to quantify chromium, cadmium, manganese, nickel and lead in human whole blood, urine, saliva and hair samples by electrothermal atomic absorption spectrometry.</a> Analytica Chimica Acta. 659 (1), 60-67 (2010).</li><li>Shyamal, M., 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=Highly+Selective+Turn-On+Fluorogenic+Chemosensor+for+Robust+Quantification+of+Zn(II)+Based+on+Aggregation+Induced+Emission+Enhancement+Feature.">Highly Selective Turn-On Fluorogenic Chemosensor for Robust Quantification of Zn(II) Based on Aggregation Induced Emission Enhancement Feature.</a> ACS Sensors. 1 (6), 739-747 (2016).</li><li>Kudo, H., Yamada, K., Watanabe, D., Suzuki, K., Citterio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-Based+Analytical+Device+for+Zinc+Ion+Quantification+in+Water+Samples+with+Power-Free+Analyte+Concentration.">Paper-Based Analytical Device for Zinc Ion Quantification in Water Samples with Power-Free Analyte Concentration.</a> Micromachines. 8 (4), 127 (2017).</li><li>Liu, R., Zhang, P., Li, H., Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-cloth+integrated+with+gravity/capillary+flow+chemiluminescence+(GCF-CL):+towards+simple,+inexpensive,+portable,+flow+system+for+measuring+trivalent+chromium+in+water.">Lab-on-cloth integrated with gravity/capillary flow chemiluminescence (GCF-CL): towards simple, inexpensive, portable, flow system for measuring trivalent chromium in water.</a> Sensors and Actuators B: Chemical. 236 (C), 35-43 (2016).</li></ol>

使用 HNB 对金属进行彩色检测提供了一种简单的方法来量化 IMAC 树脂中过渡金属离子对蛋白质污染程度。正如我们在参考文献20中所确立的，Ni2+与HNB结合，具有1：1的测结和与pH值一起变化的Ni-HNB复杂变化的解散常数。但是，对于所有建议的 （7-12） pH 值，复合 Kd处于子 nM 范围内。实际上，这意味着只要不存在像EDTA这样的其他强合合试剂，任何测试分数中的所有Ni2+都将与H...

自Porath和同事1创立以来，固定金属亲和色谱（IMAC）已成为一种选择方法，根据蛋白质与过渡金属离子（如Zn2+、Ni 2+、Cu2+和Co2+）粘结的能力，快速分离蛋白质。这是最常见的通过工程多异氨酸标记完成，现在是最常见的色谱纯化技术之一，用于分离重组蛋白2。IMAC还发现了除重组蛋白纯化以外的应用，作为分离奎诺酮、四环素、氨基糖苷、大纤维和β-乳糖的一种用途，用于食品样品分析3，并作为识别肝癌和胰腺癌的血清蛋白标记物的一个步骤。毫不奇怪，IMAC也成为分离一些本地生物能量酶6，7，8，9，10的首选方法。然而，这些纯化方法的成功实施，研究酶活性生物能量蛋白取决于是否存在从柱基质渗入渗出至渗出的可忽略的金属阳离子水平。IMAC中常用的二价金属阳离子具有已知的病理生物学意义，即使在低浓度11，12。这些金属的生理效应在生物能量系统中最为明显，在生物能量系统中，它们可证明是细胞呼吸或光合作用的抑制剂，其作用是13、14、15。对于大多数蛋白质类别，残余污染物金属可能会干扰蛋白质的生物功能或生物化学和生物物理技术的特征，这些问题是不可避免的。
虽然氧化条件下的金属污染水平和使用伊米达索作为乙酰胺通常低16，但蛋白质分离在存在半胱氨酸还原剂（DTT，β-甲基苯丙胺等）或与强包剂（如组氨酸17，18或乙烯二甲酸（EDTA））时，会导致金属污染水平高得多19，20。同样，由于 IMAC 树脂中的金属离子经常由卡盒组协调，因此在酸性条件下进行的蛋白质洗脱也可能具有高得多的金属污染水平。溶液中的金属含量可以使用原子吸收光谱（AAS）和电感耦合等离子体质谱（ICP-MS）在ppb-ppt范围内的检测极限范围21、22、23、24进行评估。不幸的是，在传统的生物化学实验室中，AAS和ICP-MS不是实际的检测手段，因为这些方法需要获得专门的设备和培训。
此前由英国人25、26日研究过羟基苯甲酸蓝（HNB）的用途，以识别溶液中过渡金属的存在。然而，数据20中存在一些内部矛盾，这些工作未能提供适当的协议。Temel等人27和费雷拉等人28日的研究扩大了英国人与HNB合作作为潜在金属指标的研究。然而，Temel开发了一种利用AAS进行样品分析的协议，将海布仅用作包合剂。费雷拉的研究使用563nm的HNB吸收光谱的变化，这是自由染料HNB光谱的一个区域，在pH5.7时与HNB-金属复合物的光谱严重重叠，使得测定灵敏度相当低，并造成相对较弱的金属结合亲和力20。为了解决我们实验室中来自 IMAC 的 Ni2+浸出问题，我们扩展了 Brittain25、26和 Ferreria28所做的工作，以开发能够检测几种过渡金属纳米摩尔水平的易检测。我们发现，HNB将镍和其他常见的IMAC金属与亚纳米摩尔结合亲和力结合，并在PH值20的较宽范围内形成1：1复合物。此处报告的测定基于这些发现，利用HNB光谱在647nm处的吸收变化进行金属定量。该测定可在生理pH范围内使用典型生物化学实验室中常见的缓冲液和仪器进行，使用金属染料复合物的色度检测和定量，以及自由染料与金属结合时吸收率的相关变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2xYT broth</td>
			<td>Fisher Scientific</td>
			<td>BP9743-500</td>
			<td>media for E.coli growth</td>
		</tr>
		<tr>
			<td>HEPES, free acid</td>
			<td>BioBasic</td>
			<td>HB0264</td>
			<td>alternative buffer</td>
		</tr>
		<tr>
			<td>HisPur Ni-NTA resin</td>
			<td>Thermo Scientific</td>
			<td>88222</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxynaphthol blue disoidum salt</td>
			<td>Sigma-Aldrich</td>
			<td>219916-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Fisher Scientific</td>
			<td>O3196-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>BioBasic</td>
			<td>IB0277</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS, free acid</td>
			<td>BioBasic</td>
			<td>MB0360</td>
			<td>alternative buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate</td>
			<td>Fisher Scientific</td>
			<td>S369-500</td>
			<td>alternative buffer</td>
		</tr>
		<tr>
			<td>Tricine</td>
			<td>Gold Bio</td>
			<td>T870-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of metal leaching in immobilized metal affinity chromatography

酶与从固定金属亲和色谱谱（IMAC）柱中浸出的金属的污染是酶学家的主要关切，因为IMAC树脂中使用的许多常见的二价和三价阳离子对酶有抑制作用。然而，金属浸出的程度以及各种浸出和还原试剂的影响，在很大程度上，由于缺乏使用通常可用的设备的简单和实用的过渡金属定量协议，人们不太了解生物化学实验室。为了解决这个问题，我们开发了一种协议，以快速量化使用IMAC作为净化步骤制备的样品中的金属污染量。该方法使用羟基纳普霍尔蓝色（HNB）作为样品溶液中金属阳离子含量的色度指标，而UV-Vis光谱法则根据647nm的HNB光谱变化，量化纳米摩尔范围内的金属含量。虽然溶液中的金属含量历来是使用原子吸收光谱或电感耦合等离子体技术确定的，但这些方法需要在典型的生物化学实验室范围之外进行专门的设备和培训。这里提出的方法为生物化学家提供了一种简单快捷的方法，即使用现有设备和知识在不牺牲准确性的情况下确定样品的金属含量。

图2显示了在中性pH（黑线）处的自由HNB光谱和从MS1E3D129分离中测定为Ni2+的代表性分数谱。与HNB控制相比，成功的测定系列应表明647nm的吸收率降低，这与在过渡金属存在的情况下形成HNB复合物相对应。在 647 nm 处的吸收率增加将表明测定失败。或者，从 647 nm 的初始吸光度降低 90% 以上，表明金属含量过高，需要测定更多稀释分数。自由 HNB 控制没有...

Watch this Scientific Journal Video about Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography at JoVE.com

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

我们提出了一种检测方法，用于对使用固定金属亲和色谱法制备的样品引入的金属进行易定量。该方法使用羟基纳普霍尔蓝色作为色度金属指示器，使用 UV-Vis 分光光度计作为探测器。

金属浸出在固定金属亲和力色谱中的定量

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

我们的方法非常重要，因为它允许普通生物化学家快速确定使用固定金属亲和力色谱分离的样品是否受到过渡金属的污染。主要优点是易于进行测定。该检测使用大多数生物化学实验室常见的仪器和技术，可以轻松、快速地实施。在这篇文章中，我们应用这种方法与镍-NTA柱分离的样品，该方法可用于任何其他金属亲和力树脂。首次使用已知浓度的镍库存溶液时，用户应尝试此方法。这将熟悉光谱变化中显示的工作流程、样品颜色和树脂。演示这个程序的将是科尔·斯温，一个来自我实验室的技术人员。首先，打开 UV-Vis 分光光度计并预热。确定使用二极管阵列UV-Vis分光光度计测定的色谱分数，光学吸收度为280纳米，以量化蛋白质。获得 10 至 100 毫摩尔样品缓冲液，PH 介于 7 和 12 之间，例如 Tris、HEPES、MOPS 和磷酸盐缓冲液。使用每毫升库存溶液的 120 毫克 HNB 试剂，在样品缓冲液中制备 12% 的 HNB 分散量溶液。设置分光光度计以在 647 纳米时收集数据。使用填充样品缓冲液的石英库将分光光度计空白。在含有每毫升总测定量的 50 微升 HNB 库存的 cuvette 中准备一个控制解决方案。在室温下，允许控制至少孵育三分钟。测量并记录控制样品的 647 纳米的吸光度。通过将 150 微升 HNB 库存与 2850 微升适当稀释的蛋白质馏分与样品缓冲液混合，准备检测样品。在室温下，让样品孵育至少三分钟。对要测量的每个分数重复频谱记录。在获得稀释不足的情况下，647纳米的整个光谱带消失。虽然稀释过强，但样品与控制物不易使用。要确定每个样品中金属的浓度，请首先从 HNB 控制中找出每个样品吸收率在 647 纳米时的差异。使用DDF是测定分数的稀释因子的公式确定微摩尔中的金属浓度，增量 A-B-S 647 是 647 纳米的吸收度变化，3.65 倍 10 到负 2 表示 HNB 的消光系数，l 是 cuvette 的光路（以厘米为单位）。在这项研究中，此处显示了从MSP1E3D1分离中对镍离子进行分谱的中性PH的免费HNB光谱。与 HNB 控制相比，647 纳米的吸收度有所下降，这与在过渡金属存在的情况下形成 HNB 复合物相对应。为了证明该测定方法的应用，分析了两种他标记的膜基架蛋白，MSP1E3D1，MSP2N2，以及一种新型的三血、c型细胞色素GSU0105，来自细菌硫化物。GSU0105中每个分数的蛋白质和镍离子含量彼此显著变化，而含有最多蛋白质的MSP1E3D1和MSP2N2的含量也具有最高的镍含量。还说明，金属含量可能无法均匀地分布在使用动员的金属亲和力色谱收集的馏分中。最重要的是，在所有样品中充分且一致地混合空白，并允许所有样品中的空白有相等的孵育时间。可以通过原子吸收光谱学或ICPMS进一步分析特殊感兴趣的蛋白质分数，以确认金属污染，并测试溷合或强结合蛋白金属离子。除了过渡金属浸出检测外，该技术还可用于测量过渡金属离子与蛋白质的结合亲和力。HNB 和样品中任何镍分别是眼睛和皮肤的刺激物。方法期间应使用标准 PPE，包括手套和眼睛保护。

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Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

抗癌金属配合物的合成与细胞毒性评价MTT法

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

科研

化学

Synthesis and Characterization of Functionalized Metal-organic Frameworks

功能化的合成与表征金属 - 有机骨架

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

Synthesis of a Water-soluble MetalÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œOrganic Complex Array

一个水溶性金属 - 有机络合物阵列合成

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

配体介导成核与钯金属纳米粒子的生长

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

Surface Functionalization of Metal-Organic Frameworks for Improved Moisture Resistance

金属-有机框架的表面功能化改善防潮性能

Metal-organic frameworks (MOFs) are a class of porous inorganic materials with promising properties in gas storage and separation, catalysis and sensing. ...

Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

超薄高性能金属-有机骨架膜的电泳结晶

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and ...

Molten-Salt Synthesis of Complex Metal Oxide Nanoparticles

复合金属氧化物纳米粒子的熔融盐合成

The development of feasible synthesis methods is critical for the successful exploration of novel properties and potential applications of nanomaterials. ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides