共查询到19条相似文献,搜索用时 593 毫秒
1.
2.
3.
4.
通过聚合物原位聚合反应,制备了部分填充的毛细管整体柱。pH 3~10的载体两性电解质被固化在该毛细管整体柱上。在引入八通进样阀、三通阀和四通连接单元的基础上,构建了适用于固化pH梯度毛细管等电聚焦整体柱(M-IPG)的平台。在蛋白质药物测定过程中,用M-IPG柱和羟丙基纤维素(HPC)涂层毛细管柱同时对曲托珠单抗和依那西谱的等电点进行了测定。结果表明,两种等电聚焦柱都能够同时分离混合蛋白质样品并测定蛋白质类药物中单抗和融合蛋白质的等电点(pI),M-IPG柱所测的pI值与HPC涂层毛细管柱测定的结果基本一致,表明了该柱在进一步构建多维分离平台进行蛋白质组学研究方面的潜力。 相似文献
5.
新型毛细管等电聚焦驱动方法的建立及其应用 总被引:2,自引:0,他引:2
毛细管等电聚焦(CIEF)在生物分离分析领域中的发展迅速.CIEF可分为两步法聚焦和一步法聚焦.电渗流(EOF)可作为一种新的驱动力.Rassi等根据串连体系中电渗流速率将被平均化的原理,将未涂层毛细管与聚乙二醇涂层的毛细管通过聚四氟乙烯管耦合起来,成功地利用毛细管区带电泳快速分离蛋白.此外,利用电渗流输液原理设计的电渗泵还可用作流动注射和微柱液相色谱的驱动系统。 相似文献
6.
采用甲基丙烯酸缩水甘油酯、丙稀酰胺为单体, N,N-亚甲基双丙稀酰胺为交联剂, 十二醇、1,4-丁二醇和DMSO为制孔剂在毛细管中合成含有环氧基的整体柱, 通过化学键合的方法将两性电解质(Ampholine)按照其等电点的顺序固定至整体柱中, 制成新型固定化pH梯度整体柱(monolithic immobilized pH gradient, M-IPG). 以四种蛋白混合物对这种整体柱在毛细管等电聚焦中的应用加以评价, 说明了M-IPG对蛋白质的分离特征. 与先前研究的聚丙烯酸酯基质固定化pH梯度整体柱相不同, 这种新型整体柱具有更好的亲水性, 能够有效防止蛋白吸附, 非常适用于蛋白质等生物大分子的分离分析. 相似文献
7.
梯度加压毛细管电色谱分离蛋白质 总被引:2,自引:0,他引:2
以1.5 μm无孔硅胶颗粒(non-porous silica,NPS)为固定相,采用电压和压力联合驱动流动相,用反相梯度加压毛细管电色谱(p-CEC)在7.5 min内实现了核糖核酸酶A、细胞色素C、溶菌酶和肌红蛋白等4种蛋白质的快速、高效的分离。比较了梯度加压毛细管电色谱和微柱液相色谱(μ-HPLC)分离蛋白质的结果,同时考察了固定相、离子对试剂三氟醋酸(TFA)浓度和电压等条件对梯度加压毛细管电色谱分离蛋白质的影响。结果表明,梯度p-CEC可以通过调节电压精细调节带电溶质的保留,提高分离选择性,缩短分离时间,得到较高的柱效。该方法在蛋白质分离分析及蛋白质组学的研究中具有很大的应用潜力,为高效快速地分离蛋白质开辟了新的途径。 相似文献
8.
9.
芯片毛细管电泳及其在生命科学中的应用 总被引:10,自引:0,他引:10
芯片毛细管电泳 (Chip CE)技术在近几年已取得了很大的进展。本文着重介绍芯片毛细管区带电泳技术 ,对等电聚焦、等速电泳、自由溶液电泳及胶束电动色谱等其它芯片电泳模式也有所提及。讨论了芯片材料和制作技术、芯片的几何形状、样品的操作和衍生、检测及芯片毛细管电泳技术的应用 ,特别是在核酸和蛋白质的分离分析中的进展 相似文献
10.
芯片国管电泳及其在生命科学中的应用 总被引:2,自引:0,他引:2
芯片毛细管电泳(Chip-CE)技术在近几年已取得了很大的进展。本文着重介绍芯片毛细管区带电泳技术,对等电聚焦、等速电泳、自由溶液电泳及胶束电动色谱等其它芯片电泳模式也有所提及。讨论了芯片材料和制作技术、芯片的几何形状、样品的操作和衍生、检测及芯片毛细管电泳技术的应用,特别是在核酸和蛋白质的分离分析中的进展。 相似文献
11.
A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt-c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt-c with the EOPACE system is demonstrated. 相似文献
12.
The development of a new capillary zone electrophoresis (CZE) method for the determination of protein oxidation by free radicals is described. The effects of various experimental conditions, such as type of capillary, pH, buffer concentration, capillary dimensions, temperature, applied voltage, and addition of some organic solvents, were investigated. The sample injection procedure was also optimized. Target proteins investigated here (lysozyme, human serum albumin, and β-lactoglobulin A) were effectively damaged by free radicals generated in the aqueous phase from 2,2′-azobis[2-amidinopropane] (AAPH). The new method allowed global protein fragmentation to be quantified as a function of time by monitoring the decrease in peak height of the native protein. Furthermore, the protective efficacy of antioxidants was assessed by concentration-inhibition curves against protein fragmentation. The method is thus suitable to screen compounds able to protect proteins against oxidative damage. 相似文献
13.
以泰勒分散理论为基础, 首次采用动态涂层毛细管来准确和快速测定蛋白质分子的扩散系数. 相似文献
14.
Whole-column imaging capillary electrophoresis with a short capillary is discussed. A short capillary (3-6 cm) coated with either fluorocarbon or polyacrylamide was used as a separation capillary. The whole capillary was illuminated with 280 nm light, and the transmitted light was monitored by a linear charge-coupled device (CCD). For the short capillary, hydrodynamic flow caused by a subtle height difference between the anodic and cathodic reservoirs affected the sample migration in the capillary greatly. Several sample injection methods, including use of a cross connection, sealing of the capillary ends with a gel, and use of a gel-filled capillary, have been discussed. The experimental results showed that the peak height decreased and peak width increased with the electromigration distance. Therefore, higher sensitivity was obtained in a short capillary rather than a long capillary. The whole-column imaging CE with the short capillary has been applied for the study of conjugation reactions of protein cytochrome c with sodium dodecyl sulfate (SDS) and the dye Congo Red. The method has also been used for in situ monitoring of the electrophoretic protein desorption process. Our technique is a unique tool for the study of protein binding reactions and the interaction between analyte and inner wall of the capillary. 相似文献
15.
胃癌是临床常见的恶性肿瘤之一。近年来寻找肿瘤相关特异蛋白质是蛋白质组学研究的热点。本文通过考察毛细管动态涂层方法、筛分介质聚环氧乙烷(PEO)的浓度、缓冲液的pH值、分离电压、温度及荧光染料对分离效果的影响,建立了毛细管电泳-激光诱导荧光法分离胃癌组织及癌旁正常组织蛋白质的方法;通过分离检测,获得两者的蛋白质指纹图谱。经分析,两者的指纹图谱相似度达到0.8以上,差异蛋白质分子质量集中在50000~100000 Da之间,提示某些小分子蛋白质可能是和肿瘤发生相关的特异蛋白质,从而缩小了特异性分子标记物的筛选范围。病理组织学分型及蛋白质电泳峰数目的统计结果验证了该方法的可靠性。该方法具有临床应用的潜力。 相似文献
16.
Free solution capillary electrophoresis was investigated for the characterization of an M(r) 100 000 purified F(ab')2. Optimization of the experimental conditions allowed the identification of five separated peaks, suggesting the presence of isoforms which differed by only 0.2 pH unit. This heterogeneity was still detectable with 80 amol of protein. After a preparative separation by chromatofocusing, identification of each form was performed for the first time by capillary electrophoresis. A quantitative and qualitative correlation with isoelectric focusing showed that free solution capillary electrophoresis represents a sensitive method for revealing subtle differences in charge, even for large proteins. 相似文献
17.
Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresis 总被引:2,自引:0,他引:2
Nilsson M Harang V Bergström M Ohlson S Isaksson R Johansson G 《Electrophoresis》2004,25(12):1829-1836
A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using alpha-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors. 相似文献
18.
Brenner-Weiss G Kirschhöfer F Kühl B Nusser M Obst U 《Journal of chromatography. A》2003,1009(1-2):147-153
A capillary electrophoresis-electrospray ionisation time-of-flight mass spectrometry (CE-ESI-TOF-MS) method for characterisation of non-covalent protein complexes is described using a coaxial liquid sheath-flow sprayer. The CE capillary was connected to the mass spectrometer using a commercial CE-MS sprayer mounted on a ceramic holder of the ESI interface of the mass spectrometer. Using myoglobin (Mb) as an example of non-covalent protein complex, the effect on complex stability caused by organic modifiers added to the sheath liquid was analysed. Depending on the amount of methanol, either intact Mb or the apoprotein and the prosthetic heme group were detected. 相似文献
19.
A novel diazoresin/poly(N‐vinyl aminobutyric acid) covalent capillary coating for the analysis of proteins by capillary electrophoresis 下载免费PDF全文
Bing Yu Mingming Jiao Hailin Cong Xi Shu Shujing Yang 《Journal of separation science》2014,37(6):725-730
A novel method for the preparation of covalently linked capillary coatings of poly(N‐vinyl aminobutyric acid) (PVAA) obtained from hydrolyzed polyvinylpyrrolidone was demonstrated using photosensitive diazoresin (DR) as a coupling agent. A layer‐by‐layer self‐assembled film of DR and PVAA based on ionic bonding was first fabricated on the inner wall of capillary, then ionic bonding was converted into covalent bonding after treatment with UV light through a unique photochemical reaction of DR. The covalently bonded coatings suppressed protein adsorption on the inner surface of the capillary, and thus a baseline separation of lysozyme, cytochrome c, BSA, amyloglucosidase, and myoglobin was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVAA coatings, the covalently linked DR/PVAA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of the highly toxic and moisture‐sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make covalently coated capillaries for CE. 相似文献