蛋白质,凝胶电泳及其分析应用

蛋白质是重要的生物高分子，具有催化、传输、运动、防御和调节等重要生理功能，其分离、纯化和表征对理解和利用它们在生命过程中的作用具有重大理论意义和实际价值。凝胶电泳是蛋白质测定占应用最广泛和最强有力的工具。本文讨论凝胶电圾其在蛋白质分析方面的新进展。首先介绍蛋白质的性质和功能，然后讨论蛋白质试样制备方法，最后评述各种凝胶电泳分离技术及其在蛋白质人离，纯化和表征等方面的应用。     

高效液相色谱在生物医药研究中的应用第三讲蛋白质的反相高效液相色谱分离

近十年来,高效液相色谱(HPLC)在蛋白质分离纯化方面取得了很大的进展,并得到了广泛的应用。HPLC包括排阻色谱、离子交换色谱、亲和色谱、疏水反应色谱以及反相色谱等,几乎已在蛋白质分离各种传统方法中得到应用。这些方法在蛋白质分离中各有其地位,但到目前为止人们普遍认为反相高效液相色谱(RP-HPLC是分离纯化蛋白质的最有效的方法     

3D打印便携式凝胶电泳装置用于蛋白质的快速检测

随着现场分析对于快速、便携和经济型检测的需求,分析仪器的便携化和微型化备受关注。3D打印技术的不断发展,将会极大推动小型化、便携式实验设备的开发和研制。分析仪器的微型化有助于促进资源不足地区在医疗现场、食品安全和环境污染等方面的现场监测。目前,用于蛋白质分离的凝胶电泳装置多为实验室用小型化分析仪器,可用于现场快速分离蛋白质的小型化仪器尚未见报道。该研究设计加工了一款便携式凝胶电泳装置,用于蛋白质的快速分离检测。首先,通过3D打印加工的凝胶电泳装置可在实验室内方便、快捷、低成本的复制。其次,通过对预染蛋白质相对分子质量标准的分离测试,对该系统结构进行优化。优化后该凝胶电泳装置电泳槽的尺寸仅为15 mm×20 mm×17 mm,采用3D打印技术可在5 h内加工完成,耗费打印材料10 mL。正负极所用电泳缓冲液共需4 mL,所使用的25 V锂电池可实现100 h左右的工作时间。装置优化后可实现蛋白质的快速高效分离。随后,在5种常用蛋白质相对分子质量标准的分离中,该装置与商业化平板凝胶电泳分离效果相当,同时具备更快的分离速度。该研究在便携式凝胶电泳装置的开发及其在蛋白质快速分离方面取得了初步成果,但在分离完成后立即对蛋白质进行定量分析以及更多实际样品的应用方面还需要进一步研究。     

亲和色谱纯化超螺旋质粒DNA的研究进展

非病毒载体质粒DNA已被广泛应用于基因治疗和DNA疫苗,目前迫切需要开发其大规模制备和分离纯化方法。亲和色谱是一种高分辨率、高选择性的分离技术,在蛋白质、抗体、核酸等生物大分子的分离纯化方面显示了良好的应用前景。本文综述了亲和色谱技术在超螺旋质粒DNA分离纯化中的研究进展,总结了各种亲和色谱方法分离超螺旋质粒DNA的机理和优缺点,并展望了亲和纯化技术在质粒DNA生产和制备中的应用前景。     

亲和膜色谱

亲和膜色谱又称亲和膜分离，其在蛋白质的分离纯化中作为一种综合性的技术出现在80年代末。亲和膜色谱主要优点是克服了颗粒状多孔载体扩散传质阻力大的缺点，代之以对流传质，这样就可以在较低的操作压和较高的流速下对目标蛋白进行快速的分离和纯化，从而缩短操作时间、提高纯化效率。本文将就近年来亲和膜色谱及其在蛋白质分离和纯化中的应用作一综述性介绍。     

脂质体在液相色谱和毛细管电泳中的应用

脂质体具有与细胞膜相似的封闭双层结构,是接近天然生物膜的理想模型。该文综述了脂质体的制备和性质表征方法,固定脂质体色谱用于药物在脂质体膜上的吸收和蛋白质与脂质体膜的相互作用研究,脂质体毛细管电泳在药物分离、蛋白质分离和蛋白质相互作用方面的应用研究。     

碳量子点荧光成像法应用于聚丙烯酰胺凝胶电泳检测人血清蛋白质的研究

建立了碳量子点荧光成像法检测聚丙烯酰胺凝胶电泳分离蛋白质如人血清蛋白质的新方法. 通过一步绿色微波法合成的碳量子点并将其应用在聚丙烯酰胺凝胶电泳分离蛋白质的检测中, 通过醋酸-醋酸钠调节孵育溶液, 优化pH值、碳量子点的用量及孵育溶液的浓度等条件, 使碳量子点和蛋白质相结合并在365 nm的紫外灯照射下得到了清晰的人血清蛋白电泳图, 该新方法具有原料便宜易得、无污染、简单、快速、高灵敏度、低背景及高分辨率的优点, 在生物技术方面和纳米技术方面具有巨大的应用前景.     

反相液相色谱在蛋白质及多肽分离分析中的应用

反相液相色谱是一种以疏水作用为基础的色谱分离模式。由于它具有分辨率高、重复性好、回收率高等优点,在蛋白质及多肽的分离分析中得到了极为广泛的应用。本文简要介绍了反相液相色谱及其分离机理,对其在蛋白质和多肽研究中的应用如分离纯化、肽图分析、酶活测定、构象变化检测及疏水作用研究等作系统综述,并展望了反相液相色谱在这一领域的发展前景。     

槲皮素键合纳米硅胶对牛血清蛋白吸附的研究

具有生理活性的蛋白质、核酸、酶等生物大分子在维持现代人类健康方面已必不可少,在医疗和食品等领域已逐渐得到更广泛的应用,这也对蛋白质、核酸、酶等生物大分子的分离纯化、分析测定的提出了新的要求.     

电泳技术在纳米颗粒分离中的应用

合成纳米颗粒常在尺寸和形状方面具有广泛分布.在很多实验中,需要利用一定大小及形状的纳米颗粒的独特物理化学性质,因此,简便快速的纳米颗粒分离技术越来越受到诸多科学领域的重视.电泳技术以其高分辨率,被广泛用于多种生物大分子如核酸、蛋白质等的分离纯化.纳米颗粒在尺寸上与生物体中的蛋白复合物、细胞器和微生物等十分接近,考虑到带电纳米颗粒与生物分子在电场中的运动行为的相似性,运用电泳技术进行纳米颗粒的鉴定、分离和纯化是一种新的思路,并取得了良好的实验结果.本文主要介绍了琼脂糖凝胶电泳、毛细管电泳以及其他一些电泳技术在纳米颗粒分离中的研究进展.     

Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis

The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.     

基于二维凝胶电泳的蛋白质定量分析技术

基于2DE的蛋白质定量分析技术通过对不同样品蛋白质表达差异的比较，定量地解析了蛋白质的动态变化，为了解和阐明细胞蛋白质功能及活动机制等提供了重要信息。本文简单介绍了传统2DE 的“一个样品一块胶”模式的定量分析技术，指出其因为耗时、繁琐及重复性较差等，在常规2DE定量分析应用中受到了制约；阐述了近年来发展的“多样品共分离”模式的定量技术，包括荧光胶内差示电泳(DIGE)、“不同胶曝光”以及稳定同位素标记技术，这类技术因有效克服了传统技术的局限性，由此提高了定量分析的能力；分别对每种蛋白质定量技术及其优缺点作了比较和评述，并对2DE定量分析技术的未来发展方向做出了展望。     

Effective Solubilization Procedure for Analysis of Silkworm Hemolymph Proteins by Two-Dimensional Gel Electrophoresis

The outstanding capability of two-dimensional gel electrophoresis in separating all types of proteins basically depends on the efficiency of sample preparation. Sample preparation is one of the most critical steps in two-dimensional gel electrophoresis. Unfortunately, due to severe solubility, resolution of protein on gel is usually hampered, and thus, analysis remains a difficult task. However, technically several problems are generally encountered during protein extraction and isoelectric focusing. In the present investigation, we emphasized on evaluation and comparison of six different protein solubilization methods intended for resolving and analyzing silkworm hemolymph proteins by two-dimensional gel electrophoresis. Our findings revealed that the buffer composition of 8 M urea, 4 % 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM Tris base, 65 mM dithiothreitol, and 0.2 % ampholyte can effectively solubilize and yields maximum protein spots.     

Protein Extraction From Cowpea Tissues for 2-D Gel Electrophoresis and MS Analysis

A method of extraction of proteins from cowpea for two-dimensional electrophoresis is presented. This method avoids loss of protein in the course of sample preparation and results in a fraction that yields reproducible 2-DE protein patterns. The efficiency of this method was demonstrated by comparison of the patterns of protein deposition in developing and mature cowpea seeds. It is also demonstrated that without further processing of the gel piece the proteins present in the spots can be directly digested with trypsin and the peptides subjected to mass spectrometry analysis for identification. Revised: 14 July and 1 August 2005     

Miniaturization of Slab Gel Electrophoresis Using Thermal Lens Microscope and its Application to Genetic Diagnostics

Slab gel electrophoresis is the most widely used separation method for DNA fragments, proteins and carbohydrates, and miniaturization of this process is expected to provide fast, inexpensive and convenient analyses. However, two problems concerning the miniaturization of gel electrophoresis have to be solved:the separation performance and spatial resolution of the detector. We demonstrated that the separation performance was improved by using a discontinuous gel in which a concentrating gel was used to stack the sample to a sharp band, and using thermal lens microscope (TLM), which is highly sensitive and has a spatial resolution of micron level even in light scattering matrices as a gel, such sharpened separated bands were successfully detected. In this paper, we developed a miniaturized slab gel electrophoresis apparatus, demonstrated high speed separation of DNA fragments, and applied it to genetic diagnosis of coronary heart disease.     

Simple Hexagonal-Pulsed-Field Gel Electrophoresis System

Abstract

A diode array was used to isolate point electrodes in a hexagonal-pulsed-field gel electrophoresis system. The electric circuit around the electrophoresis chamber was simplified by modification. The reasonably straightmigration and good resolution of large DNA molecules were observed by the system.

Several types of pulsed-field gel electrophoresis system have been developed to get straight DNA migration^1-5. We have adopted diodes to isolate     

聚丙烯酰胺双向凝胶电泳法分析变色人发角蛋白组成

报道了一种用双向聚丙烯酰胺凝胶电泳分析经Ｓ－羧甲基化修饰的变色人发角蛋白样品的方法，以含８ｍｏ１／Ｌ脲变性凝胶电泳做第一向，ＳＤＳ－ＰＡＧＥ做第二向。不同处理的变色人发得到有差别的双向电泳图谱，即不同处理方法得到的人发蛋白质组成有差异。以此可做为人发变色处理的一个定性方法。     

Methodological Challenges of Protein Analysis in Blood Serum and Cerebrospinal Fluid by Capillary Electrophoresis

Summary High-performance capillary electrophoresis (HPCE or CE) is an ultrasensitive analytical technique with high resolving power and a wide area of applications including peptide/protein analysis. Its applicability is greatly enhanced by the short separation times, the ease of method development and the minimum sample and organic solvent requirements. Various HPCE modes have been developed for peptide/protein analysis, including capillary zone electrophoresis, micellar elektrokinetic capillary chromatography, capillary isoelectric focusing, isotachophoresis, capillary gel electrophoresis and microemulsion elektrokinetic chromatography. HPCE can easily be applied to quality control of manufacturing processes or to clinical routine for diagnostic purposes due to its potential to provide information on the identity, the purity of the samples and the quantities of the constituents. Furthermore, interactions of a peptide or a protein with other molecules can be studied by HPCE. The separation principles of the various operation modes applied to peptide/protein analysis are presented in this article. Furthermore, in order to exemplify the application of the separation principles in the area of serum protein analysis, which is of importance in clinical practice, the capillary electrophoretic methods developed for analysis of serum and cerebrospinal fluid proteins are also reviewed.Presented at: International Symposium on Separation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, The Netherlands, February 5–7, 2003     

Phosphoproteome Profiling Using a Fluorescent Phosphosensor Dye in Two-Dimensional Polyacrylamide Gel Electrophoresis

We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4–5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS.     

毛细管凝胶电泳柱技术的新进展*

全面介绍了当前毛细管凝胶电泳(CGE) 柱制备的几种技术, 初步归纳了影响凝胶柱稳定性的可能因素, 并对毛细管凝胶电泳柱技术的几个发展方向进行了概述。共引用文献33 篇。