共查询到20条相似文献,搜索用时 171 毫秒
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膜亲和色谱的现状、发展和应用 总被引:2,自引:0,他引:2
文章对膜亲和色谱的原理、特点、设备、过程和发展情况做了介绍,并对亲和膜在整个膜分离技术中的地位、所占的比重及市场预测做了述评,对膜亲和色谱与其它色谱分离技术的优缺点进行了比较。重点介绍了制备亲和膜的材料,活化方法,间隔臂和配基的种类、选择和共价键合方法,配基和配合物产生亲和作用的机理及解离过程和方法。并对膜亲和色谱在酶、蛋白质、核糖核酸等生物大分子纯化分离方面的应用情况做了述评。 相似文献
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膜亲和色谱的现状、发展和应用 总被引:3,自引:0,他引:3
文章对膜亲和色谱的原理、特点、设备、过程和发展情况做了介绍,并对亲和膜在整个膜分离技术中的地位、所占的比重及市场预测做了述评,对膜亲和色谱与其它色谱分离技术的优缺点进行了比较。重点介绍了制备亲和膜的材料,活化方法,问隔臂和配基的种类、选择和共价键合方法,配基和配合物产生亲和作用的机理及解离过程和方法。并对膜亲和色谱在酶、蛋白质、核糖核酸等生物大分子纯化分离方面的应用情况做了述评。 相似文献
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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。 相似文献
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用磺胺甲基异恶唑作基基,大孔硅胶和为基质的高效亲和色谱分离纯化胰凝蛋白酶及胰蛋白酶。20多种蛋白质和酶在该柱上无特异性吸附。 相似文献
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乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。 相似文献
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固定金属离子亲和色谱--蛋白质分离的方法、原理、特性和应用 总被引:4,自引:0,他引:4
介绍了固定金属离子亲和色谱法(IMAC)的方法原理、金属螯合柱的制备、固定金属离子与蛋白质的相互作用以及影响这些作用的因素、不同色谱条件下各种作用力对蛋白质保留值的贡献、蛋白质的洗脱原理和IMAC在蛋白质分离纯化中的应用,论述了IMAC的特点、不足、克服的方法和今后应解决的问题。 相似文献
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《Analytical letters》2012,45(3):407-415
Aptamers are oligonucleotides or peptide molecules that are able to bind to their specific target molecules with high affinity via molecular recognition. In this study, we present development of aptamer-based affinity purification for His-tagged proteins for comparison of purification efficiency with the conventional Ni2+-based affinity chromatography. Thiol-functionalized aptamers able to specifically bind to His-tag were immobilized employing two crosslinking methods onto the surface of polystyrene resins. The resulting aptamer-anchored resins were successfully applied for purification of His-tagged proteins from complex E. coli and human cell lysates, respectively, and superior or at least comparable purification results to the conventional immobilized metal affinity chromatography were obtained via one-step purification. 相似文献
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Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography 下载免费PDF全文
Juan Yang Jing Bao Junzhong Liu Shengxiang Lin Mi Sun 《Journal of separation science》2016,39(11):2050-2056
In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. 相似文献
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Dongmei Zhou Hanfa Zou Jianyi Ni Hailin Wang Li Yang Yukui Zhang 《Chromatographia》1999,50(1-2):27-34
Summary Affinity columns suitable for HPLC were prepared by immobilization of various ligands of protein A, human IgG, human IgM and
pectinase on GMA modified cellulose membrane. The adsorption capacity, affinity efficiency and activity recovery of various
IgGs on these affinity columns were measured. It was observed that the length of the coupling arm plays a very important role
in affinity efficiency, and the effect of eluent flow-rate on adsorption capacity was very small. The protein A column was
exploited for the process monitoring of dog IgG in clinical experiments on immuno-adsorption therapy. A pectinase column was
used for the determination of polygalacturonase inhibiting proteins first purified on a hydroxyapatite column. It took only
about 2.5 min for analysis at a flow-rate of 1.0 mL min−1. The high speed analysis of biopolymers could be performed at a flow rate of 6.0 mL min−1 within 15 s. Membrane affinity chromatography gives good reproducibility, high efficiency, low column-pressure and is rapid.
It can also be used for micro-scale purification of biopolymers. 相似文献
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Although the feasibility of affinity ultrafiltration was demonstrated more than 20 years ago, commercial applications have not developed due to the high cost and practical limitations of the large macroligands needed for highly selective separations. The objective of this study was to examine the use of small charged affinity ligands for protein purification by exploiting electrostatic interactions between the charged complex and an electrically-charged membrane. Experiments were performed using bovine serum albumin and ovalbumin with Cibacron Blue as the affinity ligand. Negatively charged versions of a composite regenerated cellulose membrane were generated by covalent attachment of a sulfonic acid functionality. Binding experiments were used to identify appropriate conditions for protein separations. The selectivity for the separation of BSA and ovalbumin was a function of the solution conditions, Cibacron Blue concentration, and membrane charge, with the addition of Cibacron Blue causing a 30-fold increase in selectivity. A diafiltration process was performed at the optimal conditions, giving a BSA product with a purification factor of more than 90-fold and a yield greater than 90%. These results clearly demonstrate the potential of using a small charged affinity ligand for high resolution protein separations. 相似文献
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Whitehurst CE Yao Z Murphy D Zhang M Taremi S Wojcik L Strizki JM Bracken JD Cheng CC Yang X Shipps GW Ziebell M Nickbarg E 《Combinatorial chemistry & high throughput screening》2012,15(6):473-485
Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens. 相似文献
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Design and synthesis of affinity ligands and relation of their structure with adsorption of proteins
Ye L Xu A Cheng C Zhang L Huo C Huang F Xu H Li R 《Journal of separation science》2011,34(22):3145-3150
Affinity chromatography has played an increasingly important role both in the pharmaceutical industry and academic research. In the present study, we report our preliminary investigation on the relationship between the affinity ligand structure and its adsorption to multi-protein samples. The structure of the ligands, including the size of the ring (cyclic group) and the length of the chain (linear group), has a great impact on the adsorption of ligands to proteins. Meanwhile, the functional groups that the ligands carry are also closely related to the adsorption of ligands to proteins. This research provides good guidance for the design and synthesis of affinity materials in affinity chromatography. It is also useful to other protein-ligand interaction-related research. 相似文献
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N. Amirshakhi R. N. Alyautdin A. P. Orlov A. A. Poloznikov D. A. Kuznetsov 《Russian Journal of Physical Chemistry A, Focus on Chemistry》2008,82(11):1952-1957
A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used. 相似文献