PROTEOSTAT(R) 蛋白聚集检测

来源：作者：人气：-发表时间：2023-02-28 13:33:00【大中小】

PROTEOSTAT^® 蛋白聚集检测

某些肽链在经过错误折叠后很容易形成聚集，在生物技术和制药领域，这些聚集会严重影响蛋白的生产和应用。许多以蛋白多肽为基础研发的药物具有极大的应用前景，可是蛋白因错误折叠而导致构象发生变化形成聚集，使蛋白失活，从而影响药物疗效，造成不可估计的损失。污染物导致的蛋白聚集包括宿主细胞蛋白、非蛋白微粒和蛋白的损伤形式。

基于此 Enzo Life Sciences 推出了新产品 PROTEOSTAT^® Protein Aggregation Assay，内含红色荧光信号染料，可特异性结合聚集蛋白，进行检测。具有广泛的检测范围，可有效检测可见和不可见的蛋白微粒。

◆原理

蛋白聚集严重影响以蛋白为基础研发的药物。在药物制剂中，蛋白聚集在生物活性和免疫原性方面影响效力。

蛋白聚集发生在生产过程的各个阶段包括细胞培养、纯化、生产、储存、运输等方面。制药工业希望在生物工艺中找到新的方法，可用于检测、追踪、定量分析影响蛋白聚集的因素。

近年来以冻干稳定形式存在的蛋白聚集体作为标准品，可以准确地定量检测蛋白聚集，加上新颖的只需在酶标仪里即可实验的 PROTEOSTAT^®protein aggregation assay，可对蛋白检测方法进行优化。

在这篇文章中，使用已知浓度的聚集 IgG 标准品作为标准曲线，通过 PROTEOSTAT^® protein aggregation assay/standards 对 IgG 的聚集水平进行检测，集中分析了制药工业中常见的３种聚集形式。

　　● 热诱导聚集

　　● 机械诱导聚集

　　● 冷冻和反复冻融诱导聚集

◆优点·特色

　●　高通量筛选方法用于流式细胞仪平台监测蛋白稳定性

　●　在荧光微孔板中即可进行简单、灵敏、均一地检测

　●　不需分离目的蛋白或稀释样品

　●　与传统蛋白聚集检测染料相比更灵敏，信号更明亮

　●　优化的缓冲液和辅料用于蛋白制剂研发

　●　检测灵敏度可低至亚微摩尔级别，含量在１%-５%的聚集蛋白也能被检测出

　●　在广泛的 PH（4-10）和离子强度范围都可使用，提供至少两个数量级的线性动态范围

　●　使用 PROTEOSTAT^® 蛋白聚集检测标准品可对溶液中的聚集蛋白精确定量

　●　可检测蛋白多肽聚集程度，可用于筛选蛋白聚集激活剂或抑制剂

◆内容·使用

试剂盒包含：

PROTEOSTAT®检测试剂

PROTEOSTAT®阳性对照

PROTEOSTAT®阴性对照

10X PROTEOSTAT®分析缓冲液

在蛋白聚集实验研究中，将 PROTEOSTAT^® 蛋白聚集检测试剂盒和 Synergy Mx 多模式微孔板读数器联用，适用于监测由温度、机械损伤和反复冻融所引起的蛋白聚集。

稳定的蛋白颗粒作为标准品可快速建立标准曲线，加快定量分析蛋白聚集响应。这个检测方法能够可靠地检测到浓缩抗体制剂中小于 0.2% 的聚集蛋白，线性动态范围为２个数量级。

PROTEOSTAT^® 蛋白聚集检测试剂盒和 PROTEOSTAT^® 蛋白聚集检测标准品检测蛋白聚集特点在于操作简单，能够检测低水平的蛋白聚集情况，只需 30 分钟，就能够得到完整的分析结果。

案例一不同搅拌速率对蛋白聚集的影响	案例二蛋氨酸被氧化后可抑制蛋白聚集
使用本产品检测后，可知搅拌速度越快，蛋白聚集程度越大。	蓝色曲线为对照，没有搅拌速度在 300 rpm 下，绿色曲线与红色曲线分别表示蛋氨酸被氧化后的蛋白聚集情况与天然蛋白的聚集情况。使用本产品检测后，可知蛋氨酸被氧化后可抑制蛋白聚集。

使用说明：

应用：流式细胞术、荧光显微镜、荧光检测和微孔板
该试剂盒专门设计用于监测溶液中蛋白质聚集体的形成。

质量控制：PROTEOSTAT®蛋白质聚集检测试剂盒用于测定（1）20μM聚集溶菌酶，（2）20μM天然溶菌酶；和（3）5%聚集混合物。Z’因子大于0.5，5%的聚集信号大于无聚集质控品的3个标准方差以上。

检测数量：
ENZ-51023-KP050：在96孔板中进行50次测试，或流式细胞仪测试16次
ENZ-51023-KP002: 96孔测试2次或流式细胞仪测试70次

储存：在适当的储存条件下，试剂盒组分在产品标签注明的日期前稳定。将试剂盒储存在-20°C的无霜冰箱中，或-80°C的条件下长期储存。

运输：干冰运输

监管状态：RUO - 仅供研究用途

◆参考文献

Exposure of α-Synuclein Aggregates to Organotypic Slice Cultures Recapitulates Key Molecular Features of Parkinson's Disease: S. Moudio, et al.; Front. Neurol. 13, 826102 (2022);
GrpEL1 regulates mitochondrial unfolded protein response after experimental subarachnoid hemorrhage in vivo and in vitro: C. Ma, et al.; Brain Res. Bull. 181, 97 (2022);
Identification of an endoplasmic reticulum proteostasis modulator that enhances insulin production in pancreatic β cells: M. Miyake, et al.; Cell Chem. Biol. 1016, S2451 (2022);
Protocol for isolation and proteostatic analysis of sub-populations of spermatogenic cells in mouse: Q. Zou, et al.; STAR Protoc. 3, 101398 (2022);
ATP-citrate lyase promotes axonal transport across species: A. Even, et al.; Nat. Commun. 12, 5878 (2021);
Cell-Instructive Surface Gradients of Photoresponsive Amyloid-like Fibrils: A.M. Bender, et al.; ACS Biomater. Sci. Eng. 7, 4798 (2021);
Proteostasis regulated by testis-specific ribosomal protein RPL39L maintains mouse spermatogenesis: Q. Zou, et al.; iScience 24, 103396 (2021);
Rejuvenation of tumour-specific T cells through bispecific antibodies targeting PD-L1 on dendritic cells: L. Liu, et al.; Nat. Biomed. Eng. 5, 1261 (2021);
Characterization of Protein Aggregates, Silicone Oil Droplets, and Protein-Silicone Interactions using Imaging Flow Cytometry: C. Probst; J. Pharm. Sci. 109, 364 (2020);
Endoplasmic reticulum chaperone calmegin is upregulated in aldosterone-producing adenoma and associates with aldosterone production: K. Itcho, et al.; Hypertension 75, 492 (2020);
Monitoring plasma protein aggregation during aging using conformation-specific antibodies and FTIR spectroscopy: S. Magalhaes, et al.; Clin. Chim. Acta 502, 25 (2020), Application(s): Protein aggregation in human plasma samples;
PERK participates in cardiac valve development via fatty acid oxidation and endocardial-mesenchymal transformation: T. Shimizu, et al.; Sci. Rep. 10, 20094 (2020);
The polyphenol quercetin protects from glucotoxicity depending on the aggresome in Caenorhabditis elegans: M. Civelek, et al.; Eur. J. Nutr. 59, 485 (2020), Application(s): Protein aggregation in solution;
Fibril formation and therapeutic targeting of amyloid-like structures in a yeast model of adenine accumulation: D. Laor, et al.; Nat. Commun. 10, 62 (2019);
Transthyretin amyloid fibril disrupting activities of extracts and fractions from Juglans mandshurica maxim. var. cordiformis (Makino) kitam: N. Chaudhary, et al.; Molecules 24, 500 (2019), Application(s): Aged amyloid fibrils;
Caffeic acid and resveratrol ameliorate cellular damage in cell and Drosophila models of spinocerebellar ataxia type 3 through upregulation of Nrf2 pathway: Y.L. Wu, et al.; Free Radic. Biol. Med. 115, 309 (2018);
Discovering putative prion-like proteins in Plasmodium falciparum: A computational and experimental analysis: I. Pallares, et al.; Front. Microbiol. 9, 1737 (2018), Application(s): P. falciparum culture;
High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants: S.M. McClure, et al.; Pharm. Res. 35, 81 (2018);
The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation: M.H. Nawaz, et al.; BioSci. Rep. 38, BSR20171666 (2018), Application(s): Aggregation propensity assessed by DSF;
Aggregated transthyretin is specifically packaged into placental nano-vesicles in preeclampsia: M. Tong, et al.; Sci. Rep. 7, 6694 (2017), Application(s): Protein aggregation in extracts from placental extracellular vesicles;
Curcumin Improves Palmitate-Induced Insulin Resistance in Human Umbilical Vein Endothelial Cells by Maintaining Proteostasis in Endoplasmic Reticulum: M. Ye, et al.; Front. Pharmacol. 8, 148 (2017);
Scaffold requirements for periodontal regeneration with enamel matrix derivative proteins: A. Apicella, et al.; Colloids Surf. B Biointerfaces 156, 221 (2017), Application(s): Aggregation of enaml matrix derivative (EMD) proteins;
Trans ε-viniferin is an amyloid-β disaggregating and anti-inflammatory drug in a mouse primary cellular model of Alzheimer's disease: E. Vion, et al.; Mol. Cell. Neurosci. 88, 1 (2017); ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation: J.H. Seo, et al.; Nat. Commun. 7, 12882 (2016), Application(s): ;
BRG1 and BRM SWI/SNF ATPases redundantly maintain cardiomyocyte homeostasis by regulating cardiomyocyte mitophagy and mitochondrial dynamics in vivo: S.J. Bultman, et al.; Cardiovasc. Pathol. 25, 258 (2016);
Cardiomyocyte-specific human Bcl2-associated anthanogene 3 P209L expression induces mitochondrial fragmentation, Bcl2-associated anthanogene 3: M.T. Quintana, et al.; Am. J. Pathol. 186, 1989 (2016), Application(s): Lysates from cardiac tissues;
Interfacial dilatational deformation accelerates particle formation in monoclonal antibody solutions: G.L. Lin, et al.; Soft Matter 12, 3293 (2016);
Resveratrol reduces amyloid-beta (Aβ1–42)-induced paralysis through targeting proteostasis in an Alzheimer model of Caenorhabditis elegans: C. Regitz, et al.; Eur. J. Nutr. 55, 741 (2016), Application(s): Protein aggregation in solution;
A screening methodology for purifying proteins with aggregation problems: M. Lebendiker, et al.; Methods Mol. Biol. 1258, 261 (2015);
Effects of tau domain-specific antibodies and intravenous immunoglobulin on tau aggregation and aggregate degradation: J.O. Esteves-Villanueva, et al.; Biochemistry 54, 293 (2015);
Metal-mediated protein oxidation: applications of a modified ELISA-based carbonyl detection assay for complex proteins: H. Uehara, et al.; Pharm. Res. 32, 691 (2015), Application(s): Effects of metal-mediated protein oxidation on aggregation;
WALTZ-DB: a benchmark database of amyloidogenic hexapeptides: J. Beerten, et al.; Bioinformatics 31, 1698 (2015);
Amyloid-beta (Aβ1-42)-induced paralysis in Caenorhabditis elegans is reduced by restricted cholesterol supply: C. Regitz, et al.; Neurosci. Lett. 576, 93 (2014);
Human Stefin B Role in Cell's Response to Misfolded Proteins and Autophagy: M. Polajnar, et al.; PLoS One 9, e102500 (2014), Application(s): Proteins aggregation and oxidative stress in stefin B KO astrocytes;
Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils: A. Tsujimura, et al.; Neurobiol. Dis. 73C, 244 (2014), Application(s): In vitro formation of a-synuclein fibrils and monitoring with ProteoStat? dye;
ProteoStat to detect and discriminate intracellular amyloid-like aggregates in Escherichia coli: S.Navarro & S.Ventura; Biotechnol. J 9, 1259 (2014);
Increased carbonylation, protein aggregation and apoptosis in the spinal cord of mice with experimental autoimmune encephalomyelitis: A. Dasgupta, et al.; ASN Neuro 5, e00111 (2013), Application(s): Detection of protein aggregation in tissue section using fluorescence microscopy;
Optimization of protein purification and characterization using Thermofluor screens: S. Boivin, et al.; Protein Expr. Pur. 91, 192 (2013);
Protein quality control acts on folding intermediates to shape the effects of mutations on organismal fitness: S. Bershtein, et al.; Mol. Cell. 49, 133 (2013), Application(s): Propensity to aggregate for dihydrofolate reductase mutants;
Correction of both NBD1 energetics and domain interface is required to restore ΔF508 CFTR folding and function: W.M. Rabeh, et al.; Cell 148, 150 (2012), Application(s): Aggregation of multi-domain protein: NBD1;
Detection of α-synuclein amyloidogenic aggregates in vitro and in cells using light-switching dipyridophenazine ruthenium(II) complexes: N.P. Cook, et al.; J. Am. Chem. Soc. 134, 20776 (2012);
p62/SQSTM1-Dependent Autophagy of Lewy Body-Like α-Synuclein Inclusions: Y. Watanabe, et al.; PLoS One 7, e52868 (2012), Application(s): Protein aggregation of alpha-synuclein;
Raster image correlation spectroscopy as a novel tool for the quantitative assessment of protein diffusional behaviour in solution: Z. Hamrang, et al.; J. Pharm. Sci. 101, 2082 (2012);
High sensitivity luminescence nanoparticle assay for the detection of protein aggregation: S. Pihlasalo, et al.; Anal. Chem. 83, 1163 (2011);

◆使用流式细胞仪结合PROTEOSTAT^®蛋白聚集分析试剂盒使用例

A PROTEOSTAT^®蛋白聚集分析试剂盒检测 IgG 聚集标准品 B PROTEOSTAT^®蛋白聚集分析试剂盒检测硅油滴 C PROTEOSTAT^®蛋白聚集分析试剂盒检测 IgG 聚集标准品和硅油滴的混合物。

使用流式细胞仪结合 PROTEOSTAT^®蛋白聚集分析试剂盒检测 IgG 聚集标准品，可与其他非蛋白微粒如油滴区别开来，且无须考虑样品微粒大小（经检测 >97% 的 IgG 聚集标准品都小于２μm）。

产品编号	产品名称	产品规格
ENZ-51023-KP050	PROTEOSTAT^® 蛋白聚集检测 PROTEOSTAT^® Protein aggregation assay	50 tests
ENZ-51023-KP002	PROTEOSTAT^® 蛋白聚集检测 PROTEOSTAT^® Protein aggregation assay	2×96 tests

◆相关产品

产品编号	产品名称	包装
ENZ-51039-KP002	PROTEOSTAT^® 蛋白质聚集标准品套装 PROTEOSTAT^® Protein aggregation standards	2x96 wells

◆相关产品

人IgG多抗

◆产品详情

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浓度	1mg/ml
来源	兔
免疫原	人免疫球蛋白G
物种反应性	人
应用	WB
纯化细节	蛋白A亲和纯化
形式	液体。溶于含有10ppm卡松防腐剂的PBS缓冲液中。
使用/稳定性	在4°C下可稳定1个月，在-20°C下可稳定至少12个月。
处理	避免反复冻融
运输	冰袋运输
短期储存	+4°C
长期储存	-20°C
监管状态	RUO - 仅供研究使用

◆产品文献参考

Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation.; S. Zwicker, et al.; PLoS One 12, e0175153 (2017)

◆产品列表

产品编号	产品名称	包装
ENZ-ABS264-1000	人IgG多抗 IgG (human) polyclonal antibody	1 ml

◆相关产品

PROTEOSTAT® Protein aggregation assay PROTEOSTAT®蛋白聚集分析试剂盒
用于可见及亚可见颗粒蛋白质聚集的定量检测
ENZ-51023-KP002	96 tests×2
ENZ-51023-KP050	50 tests
PROTEOSTAT® Protein aggregation standards PROTEOSTAT®蛋白聚集检测标准品
市售蛋白质聚集测定标准品
ENZ-51039-KP002	2x96 wells
PROTEOSTAT® Thermal shift stability assay kit PROTEOSTAT®热转移稳定性测定试剂盒
用于与pH、离子强度和浓度关联的蛋白质稳定性快速筛选。
ENZ-51027-K400	400 tests
ENZ-51027-K100	100 tests
PROTEOSTAT® Protein refolding and aggregation sensing kit PROTEOSTAT® 蛋白质重折叠和聚集敏感试剂盒
用于确定蛋白质重折叠较适宜条件和功能蛋白较大回收率
ENZ-51040-KP002	2x96 wells
Protein A ELISA kit 蛋白A ELISA试剂盒
灵敏的监测污染残留用比色法酶联免疫试剂盒（9.01 pg/ml）
ADI-900-057	96 wells
E. coli host cell protein ELISA kit 大肠杆菌宿主细胞蛋白ELISA试剂盒
灵敏的定量检测大批量大肠杆菌系表达产品的宿主细胞蛋白污染(30 ng/ml)
ENZ-KIT127-0001	96 wells
E. coli host cell protein standards 大肠杆菌宿主细胞蛋白标准品
用于蛋白质印迹内部对照或ELISA的定量分析的蛋白质标准。
ENZ-PRT121-0050	50 μg
E. coli host cell proteins polyclonal antibody 大肠杆菌宿主细胞蛋白多抗
ENZ-ABS262-0100	100 μg
E. coli host cell proteins polyclonal antibody (biotin conjugate) 大肠杆菌宿主细胞蛋白多抗（生物素标记）
ENZ-ABS263-0100	100 μg
CHO host cell protein ELISA kit CHO宿主细胞蛋白ELISA试剂盒
灵敏的定量检测大批量中国仓鼠（CHO）系表达产品的宿主细胞蛋白污染(10 ng/ml)
ENZ-KIT128-0001	96 wells
CHO host cell protein standards CHO 宿主细胞蛋白标准品
用于蛋白质印迹内部对照或ELISA的定量分析的蛋白质标准。
ENZ-PRT122-0050	50 μg
CHO host cell proteins polyclonal antibody CHO宿主细胞蛋白多抗
ENZ-ABS260-0100	100 μg
CHO host cell proteins polyclonal antibody (biotin conjugate) CHO宿主细胞蛋白生物素标记多抗
ENZ-ABS261-0100	100 μg
IgG (human) polyclonal antibody 人IgG多抗
ENZ-ABS264-1000	1 mL