共查询到19条相似文献,搜索用时 78 毫秒
1.
2.
3.
4.
5.
6.
7.
基于固定化pH梯度整体材料的芯片自由流等电聚焦电泳模式的构建 总被引:1,自引:0,他引:1
芯片自由流电泳对于来源稀少的重要生物样品的连续预分级和微制备具有重要的意义。本文在自由流芯片的微分离腔内,通过原位光引发聚合反应制备了聚丙烯酰胺整体材料,并进行了pH梯度的固定化,从而构建了基于固定化pH梯度整体(M-IPG)材料的芯片自由流等电聚焦模式(μFF-IEF)。利用该新型分离模式,实现了异硫氰酸荧光素(FITC)标记的最小等电点相差0.33的甘氨酸、脯氨酸和赖氨酸混合物的分离,且分离结果优于传统的μFF-IEF。实验结果表明,通过发展基于M-IPG材料的μFF-IEF模式,不仅可以避免在缓冲溶液中添加两性电解质对后续采用其他模式分离和质谱鉴定的干扰,而且可以获得较高的分离和富集能力,有望在微量样品的连续分离和制备方面发挥重要作用。 相似文献
8.
评价了cIEF-WCID检测多肽与蛋白质药物等电点的应用效果。测定标准多肽的等电点,验证了cIEF-WCID具有高的准确度和良好的重复性(相对标准偏差0.50%)。人血红蛋白的4种主要异构体实现了基线分离;甘赖胰岛素的重复测试均只检出单一特征峰(p I 5.95±0.01);比较重组人生长激素原液和成品,等电点特征峰比例差异明显,且成品有新特征峰出现;对比进口及国产贝伐单抗,发现厂家1、3与原研药基本一致,厂家1的主成分迁移规律与原研药高度一致,厂家2的重链C末端K缺失、N末端焦谷氨酸环化或脱酰胺修饰影响了电荷异质性;通过考察尿素浓度、电解质范围和聚焦时间,优化了检测重组人促卵泡素等电点条件:2 mol/L尿素,两性电解质pH 2.5~5.0与pH 3.0~10.0按1∶1混合,聚焦电压1 000 V(1 min)~1 800 V(4 min)~2 200 V(1 min)。cIEF-WCID可快速、准确测定具有电荷异质性的蛋白类药物等电点,分辨率和重复性好,尤其是可以跟踪聚焦过程中样本的迁移特征,特别适合蛋白类药物复杂电荷异质性的检测。 相似文献
9.
用于蛋白质分析的毛细管等电聚焦-电喷雾质谱接口的改进 总被引:1,自引:0,他引:1
蛋白质组学对其分析技术提出了大规模、高通量的要求 [1] .传统的等电聚焦 ( p I) -分子量 ( MW)双向电泳技术 ( 2 D- Gel)尽管在蛋白质组学研究中占有重要地位 ,但其操作繁杂、工作周期长 .Pandey等[1] 将毛细管等电聚焦 ( CIEF)与电喷雾质谱 ( ESIMS)联用 ,使得 p I和 MW两维分离鉴定技术变得简单迅速 .但 CIEF- MS的接口操作需中断高压和将毛细管阴极端插入电喷雾管 ,故引起分析蛋白质的散焦和不重现 .本工作改进了 CIEF- MS接口 ,采用毛细管阴极端和电喷雾针一体化的电喷雾接口 ,无需中断高压 ,实现了 CIEF- MS的在线联… 相似文献
10.
利用一步法和两步法毛细管等电聚焦(cIEF)方法分离测定了蛋白质和多肽的等电点(pI)。讨论了两步法等电聚焦过程所需的溶液组成、样品进样体积、聚焦电压、聚焦时间和分离条件等因素对分离效果的影响。并对一步法和两步法进行了比较。对细胞色素C、血红蛋白、肌红蛋白、转铁蛋白和牛血清白蛋白以及6种多肽的分析结果表明:一步法步骤简单,分离速度快,可测定单一组分的pI,也能快速分离混合蛋白和多肽,但分离度较差,且不能同时准确测定各组分的pI;两步法步骤复杂,分析时间较长,但能够同时分离并准确测定混合样品中各组分的pI,所测的pI值与单一组分进行测定的结果基本一致。两种方法可相互结合、互为补充,可广泛应用于两性生物微粒等电点的快速和准确测定。 相似文献
11.
This review article describes the significant recent developments in analysis of proteins by capillary electromigration (CE) methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography) during the period 2011–2015. Improvements in sample preparation, preconcentration, suppression of adsorption and control of electroosmotic flow, separations by particular CE methods, and the detection schemes used in the analysis of proteins are discussed. Innovative applications of the above CE methods for quality control of protein biopharmaceuticals, protein determination in complex biomatrices, peptide mapping of proteins, and determination of physicochemical parameters of proteins are presented. 相似文献
12.
13.
Recombinant proteins are often produced as isoforms with different kinds and amounts of post-translational modifications that alter their function. Isoelectric focusing in shallow pH gradients, less than 0.5 pH/cm, might be capable of fractionating these isoforms. The synthetic carrier ampholyte mixtures typically used to generate these pH gradients are expensive and may adversely interact with proteins. Using defined buffers instead of synthetic carrier ampholytes reduces these problems. We tested two defined buffer systems in a vortex-stabilized electrophoresis device to see if they could form shallow pH gradients useful for separating isoforms. These pH gradients were formed by pouring a two-component concentration gradient. The poured gradients were smooth, reproducible, and stable for at least 1.5 h at 5 kV. One poured gradient focused 20 mg of cytochrome c. A second poured gradient separated glucose oxidase from amyloglucosidase. The breadth of the amyloglucosidase band indicates that the shallow, poured pH gradients can only partially separate protein isoforms at 10 kV. Proteins with pI < 0.2 pH units apart will have overlapping bands in these shallow, poured pH gradients. 相似文献
14.
《Journal of separation science》2017,40(11):2498-2505
An improved preparative method based on isoelectric focusing of analytes in a cellulose‐based separation medium is described in this study. Cellulose is suspended in an aqueous solution of simple buffers, ethylene glycol, glycerol, nonionic surfactant, and colored pI markers. Water partially evaporates during focusing run and the separation takes place in an in situ generated layer of cellulose, which has a gel‐like appearance at the end of analysis. Final positions of analytes are indicated by the positions of zones of focused pI markers. Fractions, segments of the separation medium with analytes, can be simply collected by spatula and analyzed by downstream analytical methods. Good focusing ability of the new method and almost quantitative recovery of model proteins, cytochrome c and bovine serum albumin, was verified by gel electrophoresis and capillary isoelectric focusing of the collected fractions. 相似文献
15.
The four classical modes of electrophoresis of protein molecules (sodium dodecyl sulphate electrophoresis, SDS-PAGE, isoelectric focusing, IEF, and immobilized pH gradients, IPGs, two-dimensional maps, 2D, and capillary electrophoresis, CE) are here reviewed, with special emphasis on recent innovations. Thus, in the case of SDS-PAGE, a novel method, consisting in focusing SDS-protein micelles against a gradient of cationic charges grafted onto a polyacrylamide gel is presented. In the case of IEF, the recent decoding of the structure, polydispersity, molecular mass distribution and buffering properties of the soluble carrier ampholyte buffers are here discussed. In regard to two dimensional mapping, recent instrumentation for performing 2D maps in horizontal, large gel slabs (up to 30 cm × 40 cm) and in a radial format for the SDS dimension is here evaluated. Finally, in the case of CE, three major applications are presented: a thorough study of capillary IEF and of all experimental variables, a method of importance in screening of rDNA products; the possibility of running proteins and peptide separations in very acidic, amphoteric, isoelectric buffers in absence of any capillary coating; finally, the possibility of producing a facile, user friendly, covalent coating of the wall silanols via bonding of quaternarized piperazines endowed with an iodinated tail. In acidic, volatile buffers, such protein/peptide runs can be directly interfaced with mass spectrometry instrumentation. 相似文献
16.
17.
2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE. 相似文献
18.
蛋白质结构复杂,种类多样,与各种生命活动密切相关。大部分蛋白质在生物体中含量极低,对其分析检测带来极大困难。因此实现复杂生物样品中蛋白质的选择性识别与分离,对实现蛋白质的分离分析意义重大。通过分子印迹技术制备的分子印迹聚合物含有与模板分子大小、形状一致,官能团相互匹配的三维印迹空穴,在蛋白质的选择性识别与分离领域显示出了巨大的发展潜力。但是,由于蛋白质具有尺寸较大、构型易变、结构复杂等特点,分子印迹技术在蛋白质印迹中面临着巨大挑战。该文在介绍几种新型分子印迹技术包括表面印迹、抗原决定基印迹和金属螯合物印迹的基础上,综述了近3年分子印迹技术在蛋白质分离分析方面的应用,并对其发展进行了总结与展望。 相似文献