Isoelectric focusing

等电聚焦
  • 文章类型: Review
    血红蛋白的遗传调节是复杂的,并且存在导致临床上重要的血红蛋白病症的许多遗传异常。这里,我们回顾了血红蛋白疾病的分子病理生理学,并回顾了诊断这些疾病的新旧方法。及时诊断婴儿血红蛋白病对于协调最佳的挽救生命的干预措施至关重要。准确识别有害突变的携带者可以进行遗传咨询和知情的计划生育。遗传性血红蛋白疾病的初始实验室检查应包括全血细胞计数(CBC)和外周血涂片,其次是根据临床怀疑和现有方法精心选择的检查.我们讨论了分离血红蛋白的各种方法的实用性和局限性,包括醋酸纤维素和柠檬酸琼脂血红蛋白电泳,等电聚焦,高分辨率高效液相色谱,和毛细管区带电泳。认识到血红蛋白疾病的全球负担大部分存在于低收入和中等收入国家,我们回顾了越来越多可用的护理点测试(POCT),它们在扩大早期诊断计划以解决镰状细胞病的全球负担方面发挥着越来越重要的作用,包括镰刀扫描,HemoTypeSC,瞪羚Hb变体,智能生活LC全面了解血红蛋白和珠蛋白基因的分子病理生理学,以及清楚了解当前可用的诊断测试的实用性和局限性,对于减少全球疾病负担至关重要。
    The genetic regulation of hemoglobin is complex and there are a number of genetic abnormalities that result in clinically important hemoglobin disorders. Here, we review the molecular pathophysiology of hemoglobin disorders and review both old and new methods of diagnosing these disorders. Timely diagnosis of hemoglobinopathies in infants is essential to coordinate optimal life-saving interventions, and accurate identification of carriers of deleterious mutations allows for genetic counseling and informed family planning. The initial laboratory workup of inherited disorders of hemoglobin should include a complete blood count (CBC) and peripheral blood smear, followed by carefully selected tests based on clinical suspicion and available methodology. We discuss the utility and limitations of the various methodologies to fractionate hemoglobin, including cellulose acetate and citrate agar hemoglobin electrophoresis, isoelectric focusing, high-resolution high-performance liquid chromatography, and capillary zone electrophoresis. Recognizing that most of the global burden of hemoglobin disorders exists in low- and middle-income countries, we review the increasingly available array of point-of-care-tests (POCT), which have an increasingly important role in expanding early diagnosis programs to address the global burden of sickle cell disease, including Sickle SCAN, HemoTypeSC, Gazelle Hb Variant, and Smart LifeLC. A comprehensive understanding of the molecular pathophysiology of hemoglobin and the globin genes, as well as a clear understanding of the utility and limitations of currently available diagnostic tests, is essential in reducing global disease burden.
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  • 文章类型: Journal Article
    这篇综述全面概述了过去二十年来CE在重组治疗和诊断蛋白的分析和表征中的方法学进展和应用。综述的第一部分讨论了生物技术蛋白质生产的各个方面以及对最终产品的相关影响。这包括上游流程,例如,选择和转染宿主细胞,扩大细胞培养和培养条件,以及下游加工和生物技术制造未来趋势的讨论。这一部分对于将生物技术生产与分析挑战和要求联系起来,以便提供全面的见解至关重要。在这种情况下,生产步骤对最终药物/产品的质量的影响将在相关的目标分子的翻译后修饰方面进行讨论,主要关注糖基化模式和构象效应.特别注意影响重组产物的功效和安全性的宿主细胞特异性和非人修饰。被赋予了这种预言性的知识,综述的主要部分讨论了不同CE技术对制造过程的开发和优化的多种贡献,最终药品的评价和表征及其在质量控制中的作用。不同的CE技术,比如CZE,毛细管凝胶电泳(CGE),(成像)毛细管等电聚焦((i)CIEF),µChipCE,CE-Western印迹,亲和力CE(ACE),讨论了CE-MS,包括简要介绍了各自的分离和连字符原理,以及它们在不同重组生物制品分析中的应用以及最新策略。所解决的分析物组合包括各种重组蛋白,分子量从4.1kDa到20.3MDa(对于重组病毒样颗粒),和pI范围从2.0到11.2。调查中未明确涵盖抗体。通过编制验证方面和拟议的适用性测试来补充审查,以确保方法满足工业和制药需求的可行性。
    This review provides a comprehensive overview of methodological advances and applications of CE in the analysis and characterization of recombinant therapeutic and diagnostic proteins over the past two decades. The first part of the review discusses various aspects of biotechnological protein production and the related effects on the final product. This covers upstream processes, e.g., selection and transfection of host cells, up-scaling of cell cultures and cultivation conditions, as well as downstream processing and a discussion of future trends in biotechnological manufacturing. This part is essential for relating biotechnological production to analytical challenges and requirements in order to provide a holistic insight. In this context, the influence of manufacturing steps on the quality of the final drug substance/product is discussed in terms of related post-translational modifications of the target molecule with a major focus on glycosylation pattern and conformational effects. Particular attention is given to host cell specific and non-human modifications affecting the efficacy and safety of recombinant products. Endowed with this propaedeutic knowledge, the major part of the review discusses the manifold contributions of different CE techniques to the development and optimization of the manufacturing process, to the evaluation and characterization of the final drug product and their role in quality control. Different CE techniques, such as CZE, capillary gel electrophoresis (CGE), (imaged) capillary isoelectric focusing ((i)CIEF), µChipCE, CE-Western blot, affinity CE (ACE), and CE-MS are discussed including a brief introduction in the respective separation and hyphenation principle as well as their applications in the analysis of different recombinant biologics together with recent strategies. The addressed analyte portfolio comprises a vast variety of recombinant proteins with molecular masses from 4.1 kDa up to 20.3 MDa (for recombinant virus-like particles), and a pI range from 2.0 to 11.2. Antibodies are not explicitly covered in the survey. The review is complemented by compiling validation aspects and proposed suitability tests in order to assure the feasibility of methods to industrial and pharmaceutical needs.
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  • 文章类型: Journal Article
    本文提供了2021年毛细管电泳(CE)技术的年度综述。从ISIWebofScience检索到2021年发表的与CE技术相关的291篇研究论文,\"毛细管电泳-质谱\"\"毛细管等电聚焦\"\"胶束电动色谱\",或\"毛细管电泳\"(非\"毛细管电色谱\"\"微芯片\"和\"毛细管整体柱\")。此外,综述了中国期刊上与CE技术相关的9篇研究论文:中国色谱杂志和中国分析化学杂志。这篇综述集中在《配位化学评论》上发表的七篇论文,AngewandteChemie-国际版,自然协议,TrAC-分析化学趋势,和信号转导和影响因子(IFs)大于10.0的靶向治疗,以及在分析化学中报告的42篇论文,分析ChimicaActa,塔兰塔,和食品化学,IFs在5.0到10.0之间。这篇综述还全面概述了IFs<5.0的色谱A和电泳杂志中具有代表性的CE著作,以及重要的中文期刊,中国色谱杂志和中国分析化学杂志。根据IF,本文介绍了CE相关论文的代表性著作,让读者快速了解CE技术在过去一年的重要研究进展。
    This paper provides an annual review of capillary electrophoresis (CE) technology in 2021. A total of 291 research papers related to CE technology published in 2021 were retrieved from the ISI Web of Science using the keywords, \"capillary electrophoresis-mass spectrometry\" \"capillary isoelectric focusing\" \"micellar electrokinetic chromatography\", or \"capillary electrophoresis\" (not \"capillary electrochromatography\" \"microchip\" and \"capillary monolithic column\"). In addition, nine research papers related to CE technology in Chinese journals were reviewed: Chinese Journal of Chromatography and Chinese Journal of Analytical Chemistry. This review focused on seven papers published in Coordination Chemistry Reviews, Angewandte Chemie-International Edition, Nature Protocols, TrAC-Trends in Analytical Chemistry, and Signal Transduction and Targeted Therapy with impact factors (IFs) greater than 10.0, as well as 42 papers reported in Analytical Chemistry, Analytica Chimica Acta, Talanta, and Food Chemistry with IFs between 5.0 and 10.0. This review also provides a comprehensive overview of representative CE works in Journal of Chromatography A and Electrophoresis with IFs<5.0, as well as important Chinese journals, Chinese Journal of Chromatography and Chinese Journal of Analytical Chemistry. According to the IF, this paper introduces the representative work of CE-related papers to allow readers to quickly understand the important research progress of CE technology in the past year.
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  • 文章类型: Journal Article
    本文详细概述了2017年至2021年中期通过毛细管电迁移(CE)方法分离和分析蛋白质的相关进展。提出的主题包括样本准备,抑制蛋白质吸附,控制电渗流,通过特定的CE方法分离蛋白质以及通过各种检测技术进行蛋白质鉴定和定量。为了说明CE方法在蛋白质分析中的广泛可用性,本文展示了CE方法的几个新应用,如蛋白质生物制药的质量控制和表征,测定复杂生物基质中的蛋白质,翻译后修饰的研究,肽作图,蛋白质与其他(生物)分子的相互作用,并确定其物理化学和生化特性。
    This paper provides a detailed overview of relevant developments in separation and analysis of proteins by capillary electromigration (CE) methods in the period 2017-mid 2021. The presented topics embrace sample preparation, suppression of protein sorption, control of electroosmotic flow, separations of proteins by the particular CE methods and protein identification and quantification by various detection techniques. To illustrate the wide usability of CE methods for protein analysis, the paper shows several novel applications of CE methods, such as quality control and characterization of protein biopharmaceuticals, determination of proteins in complex biomatrices, investigation of posttranslational modification, peptide mapping, interactions of proteins with other (bio)molecules and determination of their physicochemical and biochemical characteristics.
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  • 文章类型: Journal Article
    脑脊液(CSF)中存在≥3个寡克隆带(OCB),而血清中没有相应的带代表了明确的病理模式,而1-2条CSF条带(临界模式)的临床意义研究甚少。
    我们筛选了1986年连续的CSF和血清样品,这些样品是在四年的时间内收集的,并获得了等电聚焦(IEF)的结果。对于临界OCB患者,我们回顾了各个医学图表,以评估临床诊断。在可行的情况下,重复IEF并获得后续样品的结果。使用聚丙烯酰胺凝胶进行IEF,然后进行免疫印迹和IgG特异性抗体染色。此外,我们使用不同的CSF限制带截断值对OCB的诊断特异性进行了系统文献综述.
    在253名临界OCB患者中,21.7%患有中枢神经系统炎症性神经系统疾病(IND),包括4%的多发性硬化症患者,14.2%有外周IND,而其余64.1%的患者表现为非炎症性疾病。一个或两个没有相应血清带的CSF带的频率在疾病组之间没有差异。在100名患者的亚组中重复IEF。其中,73%为OCB阴性,而没有样本是阳性的。在中位数为27个月后收集的随访样本中,有26例患者的IEF结果可用。其中,4(15.4%)转正。系统文献综述显示,在主要患有非炎性神经系统疾病的患者中,使用≥3和≥2条CSF带的OCB诊断特异性分别为97%和92%。
    一个或两个CSF限制带的临床意义是中等的,因此,表明鞘内B细胞活性的可能但不可靠的证据。样品重新测试,引入额外的诊断类别,例如“可能的鞘内IgG合成”,和后续腰椎穿刺可能是解决这种情况的可能选择。
    The presence of ≥3 oligoclonal bands (OCB) in the cerebrospinal fluid (CSF) without corresponding bands in serum represents a definite pathological pattern, whereas the clinical significance of 1-2 CSF bands (borderline pattern) is poorly investigated.
    We screened 1986 consecutive CSF and serum samples which were collected over a four-year time period and had results of isoelectric focusing (IEF) available. Of patients with borderline OCB we reviewed individual medical charts for assessment of clinical diagnoses. Where feasible, IEF was replicated and results of follow-up samples were obtained. IEF was performed using polyacrylamide gel followed by immunoblotting and IgG-specific antibody staining. Additionally, we performed a systematic literature review of the diagnostic specificity of OCB using different cut-offs for CSF-restricted bands.
    Out of 253 patients with borderline OCB, 21.7% had an inflammatory neurological disease (IND) of the central nervous system, comprising 4% multiple sclerosis patients, and 14.2% had a peripheral IND, whereas the remaining 64.1% of patients showed non-inflammatory diseases. Frequency of one or two CSF bands without corresponding serum bands did not differ between the disease groups. In a subgroup of 100 patients IEF was repeated. Of those, 73% were OCB negative, while no sample was positive. In 26 patients IEF results were available of a follow-up sample collected after a median of 27 months. Of those, 4 (15.4%) turned positive. Systematic literature review revealed a diagnostic specificity of OCB of 97% and 92% using a cut-off ≥3 and ≥2 CSF bands in patients with mainly non-inflammatory neurological diseases.
    The clinical significance of one or two CSF-restricted bands is moderate and, hence, indicates a possible but not reliable proof of intrathecal B-cell activity. Sample re-testing, introduction of an additional diagnostic category, e.g. \"possible intrathecal IgG synthesis\", and follow-up lumbar puncture might be possible options to address this scenario.
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  • 文章类型: Journal Article
    用于定性和制备目的的电泳方法通常用于生物研究中,并且在大多数研究实验室中长期以来已被确立为不可或缺的分析工具。这里列出了一些最近为特殊研究目的而开发的更专业的创新。这些包括液体悬浮液中的微制备等电聚焦,通过SDS-PAGE加速蛋白酶消化,二维SDS-PAGE分析膜蛋白,用于补体C3分辨率的碳纳米管修饰页,酸性介质中超酸性蛋白质组的电泳分辨率,和琼脂糖中前β/α脂蛋白A-I的二维免疫电泳。本章简要回顾了所有这些方法。
    Electrophoretic methodologies for qualitative and preparative purposes are commonly used in biological research and have been well established as an integral analytical tool for a long time in most research laboratories. Listed here are some of the more specialized innovations that have been developed in recent times for special purposes of study. These include micropreparative isoelectric focusing in liquid suspension, accelerated protease digestion by SDS-PAGE, two-dimensional SDS-PAGE for membrane protein resolution, carbon nanotube-modified page for resolution of complement C3, electrophoretic resolution of ultra-acidic proteomes in acidic media, and two-dimensional immunoelectrophoresis of pre-beta/alpha lipoprotein A-I in agarose. All these methods are briefly reviewed in this chapter.
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  • 文章类型: Journal Article
    与一维分离相比,根据两种不同的凝胶电泳方法分离蛋白质不仅提高了高度复杂样品的分辨率,但也是蛋白质和蛋白质复合物表征的有价值的工具。有许多不同的电泳方法可以组合。变性条件下的等电聚焦和SDS聚丙烯酰胺凝胶电泳的组合提供了所有生物分析技术的最高分辨率。这是对双向电泳方法的历史和技术水平的简短回顾,并包含一些高分辨率2D电泳的实用技巧,这是基于几十年来这种方法的经验。
    Separating proteins according to two different gel-electrophoretic methods not only increases the resolution power for highly complex samples when compared to one-dimensional separations, but is also a valuable tool for protein and protein complex characterization. There are a number of different electrophoresis methods which can be combined. The combination of isoelectric focusing under denaturing conditions and SDS polyacrylamide gel electrophoresis delivers the highest resolution of all bio-analytic techniques. This is a short review on the history and state of the art of two-dimensional electrophoresis methods, and contains some practical tips for high resolution 2D electrophoresis, which are based on several decades of experience with this method.
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  • 文章类型: Journal Article
    质谱(MS)技术通常用于蛋白质鉴定和高分辨率2-D电泳后对所选蛋白质点的进一步分析。在过去的几年中,已经开发了互补的无凝胶方法,并且已被证明是现代蛋白质组学中的有用工具。因此,用于根据pi差异进行蛋白质分级的各种无凝胶电泳装置的开发和应用是感兴趣的主题。这篇综述描述了基于Agilentoffgel3100分级分离器的等电聚焦(IEF)无凝胶电泳的当前状态。审查包括,因此,(i)有关IEF以及其他先前的IEF无凝胶电泳发展的概述;(ii)粗胶的基本原理和未来趋势;(iii)当前粗胶程序的优缺点;(iv)分离的蛋白质颗粒用于进一步粗胶分离的要求;(v)通过先进的电泳和色谱技术进行二维分析的粗胶分数要求;(vi)粗胶操作条件对金属-蛋白质复合物稳定性的影响。
    Mass spectrometry (MS) techniques are commonly used for protein identification and further analysis of selected protein spots after high resolution 2-D electrophoresis. Complementary gel-free approaches have been developed during the last few years and have shown to be useful tools in modern proteomics. The development and application of various gel-free electrophoresis devices for performing protein fractionation according to the pI differences is therefore a topic of interest. This review describes the current state of isoelectric focusing (IEF) gel-free electrophoresis based on the Agilent offgel 3100 fractionator. The review includes, therefore, (i) an overview on IEF as well as other previous IEF gel-free electrophoresis developments; (ii) offgel fundamentals and future trends; (iii) advantages and disadvantages of current offgel procedures; (iv) requirements of isolated protein pellets for further offgel fractionation; (v) offgel fraction requirements to perform the second dimensional analysis by advance electrophoresis and chromatographic techniques; and (vi) effect of the offgel operating conditions on the stability of metal-protein complexes.
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  • 文章类型: Journal Article
    用于定性和制备目的的电泳方法的使用通常用于生物研究中,并且在大多数研究实验室中长期以来已被确立为整体分析工具。这里列出了一些最近为特殊研究目的而开发的更专业的创新。这些包括液体悬浮液中的微制备等电聚焦,通过SDS-PAGE加速蛋白酶消化,二维SDS-PAGE分析膜蛋白,用于补体C3分辨率的碳纳米管修饰页,酸性介质中超酸性蛋白质组的电泳分辨率,和琼脂糖中前β/α脂蛋白A-I的二维免疫电泳。本章简要回顾了所有这些方法。
    The use of electrophoretic methodologies for use in qualitative and preparative purposes are commonly used in biological research and have been well established as an integral analytical tool for a long time in most research laboratories. Listed here are some of the more specialized innovations that have been developed in recent times for special purposes of study. These include micropreparative isoelectric focusing in liquid suspension, accelerated protease digestion by SDS-PAGE, two-dimensional SDS-PAGE for membrane protein resolution, carbon nanotube-modified page for resolution of complement C3, electrophoretic resolution of ultra-acidic proteomes in acidic media, and two-dimensional immunoelectrophoresis of pre-beta/alpha lipoprotein A-I in agarose. All these methods are briefly reviewed in this chapter.
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  • 文章类型: Journal Article
    Since 1970, a series of systematic investigations have been performed on three modes of moving reaction boundaries (MRBs), viz. moving precipitate boundary (MPB), moving neutralization boundary (MNB) and moving chelation boundary (MCB). So far, there has not been any review on them. Herein, we firstly review the MPB with respect to its velocity, product concentration, elution displacement, stacking effect, separation effect, judgment expressions and related procedures. We then make a review on the MNB about its theory, demonstration, improvement, and uses in isoelectric focusing (IEF), sample stacking, MNB-based stacking mechanisms and relevant computer simulations. We further give a review on the MCB regarding the relevant theory, verifications, and applications in EDTA-based sweeping and computer simulation. Finally, we give an outlook on the MRB study. More than 150 references are cited.
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