连续自由流电泳的研制和应用于分离蛋白质和细胞

连续自由流电泳(Continuous free-flow dectrophoresis,CFE)自1980年起有了相当大的发展,其主要特点是将只能分析少量物质(1μg)的分析型电泳转变成能分离lg左右物质的制备型电泳,以达到分离和纯化各种生物产品目的^[1]。近年来CFE广泛应用于蛋白质和细胞分离,是一种很有前途的制备级电泳方法^[2,3]。在连续自由流电泳中,一种恒定pH称为载体缓冲液的液体．沿垂直方向缓慢流过一矩形的电泳薄腔,电泳腔的两侧是电极室,能被横向地加上直流电场,欲分离的蛋白质混合物通过一根细管从样品入口处连续注入腔体,随缓冲液措径向流动,同时又向与液流垂直的方向迁移．每种蛋白质的侧移距离与它的泳动率、电场强度及它在分离腔中的保留时间成正比。在分离腔出口处,混合物中各种蛋白质根据它们的泳动率被分配在各股液流中并由腔体出口处的一组收集管收集。但是有许多次级现象能损害连续自由流电泳的分离结果,诸如沉降效应、电渗流、焦尔热和自然对流。本文报道了仪器的研和我们对模型蛋白分离条件进行了优化,并成功地将人和兔的红细胞分开。     

微芯片电泳-紫外检测系统分析蛋白质

微芯片电泳是基于微机电加工技术(MEMS)工艺,在芯片上的微管道中完成电泳检测过程的新型技术.依据紫外吸光度分析法,对蛋白质样品进行电泳分离与紫外检测.实验采用自控接口模板对进样及分离电压进行了系统的程序化控制,从而准确地控制整个电泳、检测流程,提高了微芯片电泳的分离效率和检测灵敏度.实验结果表明,夹流进样的方法可以有效分离混合蛋白,可用于蛋白质样品的分离检测.     

精编细胞生物学实验指南（Shoa Protocols in Cell Biology）

本书内容详实全面，主要包括：细胞培养、细胞的制备与分离、亚细胞组分分离和细胞器分离、抗体、显微镜技术、细胞蛋白质的特性、电泳与免疫印迹、蛋白标记和免疫沉淀、蛋白质的磷酸化作用、蛋白质转运，以及细胞增殖、衰老和死亡、体外重建、细胞黏附和细胞外基质、细胞的能动性、细胞器运动。全书还附有各种实用信息和数据，包括试剂与溶液的配制、细胞生物学研究中常用的药物概要和各种常用技术的介绍等。     

连续自由流电泳样机的应用

用连续自由流电泳(continuous flow electrophoresis,CFE)成功地将细胞色素c(Cyt c)和牛血红蛋白(Hb)两种模式蛋白分开,电泳分离后的样品经过紫外分光光度计和聚丙烯酰胺凝胶电泳检测.样品的分离结果与众多性能参数有关,如:分离室间隙,样品流速,缓冲液流量、pH、电导率及分离功耗等.     

用自由流式电泳法分离及分析不同活性状态T、B淋巴细胞

由于淋巴细胞各亚群在造血调控中占有重要的地位,分离取得纯度较高、数量较大又保持生物活性的T、B淋巴细胞亚群对于进一步研究淋巴细胞在造血中的作用是极为重要的。本实验利用自由流式电泳法,根据细胞表面所带电荷基团数量和性质的差异,分离出不同活性的淋巴细胞亚群,同时进行了某些特性的分析。     

薄膜电泳

电泳方法是对生物组成成份的分离、分析不可缺少的技术。近年来相继出现了薄膜电泳,聚丙烯酰胺凝胶电泳,等电聚焦等新的电泳技术。应用有支持物的电泳技术,不仅能鉴别和分析构成生物体的成份和生物大分子,同时对细胞的分离和纯化方面的应用也不断有发展。由于薄膜电泳所用的装置和用具可以自己制做,加上操作简便迅速,因而这样的电泳方法就成为生化研究中的常规重要技术之一。这种用支持物的电泳也叫区带电泳。薄膜电泳主要是用纤维素薄膜     

蛋白质和多肽研究的毛细管电泳技术

毛细管沈阳东大迪克化工药业有限公司电泳（capillary　electrophoresis，CE）也常称高效毛细管电泳（high performance capillary electrophoresis．HPCE）．是以内径20-200μm的柔性毛细管柱作为分离通道、以高压直流电场为驱动力对各种小分子、大分子以至细胞等进行高效分离、检测或微量制备等有关技术的总称（参见图1）。     

大蒜有丝分裂不同时期蛋白质的变化

将细胞的显微分离、微量蛋白电泳和超敏感银染有机结合起来,对显微分离的大蒜根端分生组织间期、前期、中期、后期和末期各１００个细胞进行了蛋白电泳。发现在不同时期,含量较高的蛋白谱带有７条,其中４５ｋＤ和６５ｋＤ的蛋白在间期、前期、中期、后期和末期呈周期性变化     

科学出版社生命科学编辑部新书推介

精编细胞生物学实验指南(Short Protocols in Cell Biology)[美]J.S.博尼费斯农等编章静波等译978-03-017579-4定价:120.002006年12月出版本书内容翔实全面,主要包括:细胞培养、细胞的制备与分离、亚细胞组分分离和细胞器分离、抗体、显微镜技术、细胞蛋白质的特性、电泳与免疫印迹、蛋白标记和免疫沉淀、蛋白质的磷酸化作用、蛋白质转运,以及细胞增殖、衰老和死亡、体外重建、细胞黏附和细胞外基质、细胞的能动性、细胞器运动.全书还附有各种实用信息和数据,包括试剂与溶液的配制、细胞生物学研究中常用的药物概要和各种常用技术的介绍…     

科学出版社生命科学编辑部新书推介

《精编细胞生物学实验指南》[美]J．S．博尼费斯农等编章静波等译978-03-017579-4定价:120．00元2006年12月出版本书内容主要包括:细胞培养、细胞的制备与分离、亚细胞组分分离和细胞器分离、抗体、显微镜技术、细胞蛋白质的特性、电泳与免疫印迹、蛋白标记和免疫沉淀、蛋白质的磷酸化作用、蛋白质转运,以及细胞增殖、衰老和死亡、体     

Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed.     

Free-flow electrophoresis. II. Analysis of the method with respect to preparative cell separation.

Electrophoretic cell separation by means of free-flow electrophoresis in an FF5 apparatus was investigated with respect to band resolution, separation capacity, reproducibility and influence on cell viability. Very sharp bands and a large separation capacity were achieved using triethanolamine/acetate buffered glycine media as liquid curtain. Acid buffer ions such as N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES) or phosphate produced broader bands. Osmotic expanders such as saccharides, though preserving cell viability excellently, decrease electrophoretic velocity and thus separation capacity. The decrease in cell viability observed in glycine media could be compensated for by addition of Ca2. Band broadening caused by methodologically specific velocity flow profiles could be reduced to a negligible level by coating the chamber walls with albumin and by appropriate adjustment of sample flow rate and liquid curtain velocity. Under the optimum conditions described, selective cell loss and artificial change in electrophoretic mobility of the cells during operation can be disregarded. The main reason for cell loss was cell aggregation at low ionic strength, which can be prevented or reversed by treatment of the cells with deoxyribonuclease.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

免疫磁珠法分离鼻咽癌基质的T淋巴细胞

肿瘤内环境与肿瘤的发生密切相关.肿瘤细胞周围的组织在癌变发生时不会是一个沉默的旁观者,可能在肿瘤的发生和发展中扮演十分重要的角色.本研究分别采用不同的磁珠分选技术分离T淋巴细胞.采用CK LMP1,CD105和成纤维细胞表面蛋白,结合全血总T细胞试剂盒,间接法分离鼻咽癌基质的T淋巴细胞;采用CD3直接磁分选法分离鼻咽癌基质的T淋巴细胞,然后用免疫组化法鉴定分选的效果和细胞的质量.结果表明,免疫组化显示间接磁分选法分离出来的T淋巴细胞不能完全去除肿瘤细胞,RNA的质量不佳;而直接磁分选分离出来的T淋巴细胞为纯净的T淋巴细胞,而且RNA的质量良好.提示直接磁分选技术是分离鼻咽癌基质T淋巴细胞的首选方法.     

Separation of leucocytes in the dogfish (Scyliorhinus canicula) using density gradient centrifugation and differential adhesion to glass coverslips

Summary The blood of the dogfish, S. canicula, contains several types of leucocytes, namely thrombocytes, monocytes, lymphocytes and four populations of granulocytes. Three of these granulocyte types, G1, G3 and G4, are eosinophilic while G2 is heterophilic/neutrophilic. All of the leucocyte types, with the exception of G2 granulocytes and monocytes, can be separated by means of their differential adherent properties to glass and by density gradient centrifugation. Thrombocytes, G3 and G4 granulocytes can be separated in good purity by single-step methods while G1 granulocytes and lymphocytes require a combination of density gradient centrifugation followed by adherence to glass to remove contaminating thrombocytes. Depending on the cell type, between 11–45% of cells with consistently high viability can be recovered after separation. Separated populations of the thrombocytes and granulocytes will be especially useful for studies on the role of such cell types in inflammation.     

Separation of rat leukocytes by countercurrent distribution in aqueous two-phase systems. I. Characterization of subpopulations of cells.

Cells from rat spleen, lymph nodes, and thoracic duct were separated by countercurrent distribution in aqueous two-polymer phase systems containing dextran and polyethylene glycol. Lymphoid cells from the different organs gave distinct, highly reproducible distribution patterns. The yield of separated cells and their viability compared well with other methods of physical separation. The majority of the leukocytes was separated from erythrocytes. Cells with surface immunoglobulin were recovered in one side of the distribution, while thymus-derived lymphocytes as determined by indirect immunofluorescence and histochemical staining were found in all fractions. However, cells responding to PHA and Con A were concentrated in a small area of the distribution, indicating a separation of subpopulations of thymus-derived lymphocytes.     

High gradient magnetic cell separation with MACS.

A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested. Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns. Unlabelled cells pass through the column, while labelled cells are retained. The retained cells can be easily eluted. More than 10(9) cells can be processed in about 15 min. Enrichment rates of more than 100-fold and depletion rates of several 1,000-fold can be achieved. The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry. Light scatter and fluorescent parameters of the cells are not changed by the bound particles. Magnetically separated cells can be analysed by fluorescence microscopy or flow cytometry or sorted by fluorescence-activated cell sorting without further treatment. Magnetic tagging and separation does not affect cell viability and proliferation.     

Electrophoretic mobility of lymphocytes in chronic lymphatic leukemia (author's transl)]

Lymphocytes and other blood cells can be separated by means of free flow cell electrophoresis. Immunofluorescence of the separated lymphocytes of four healthy volunteers with antiimmunoglobulins IgD and IgM produced different distribution profiles for each immunoglobulin class, the IgD positive cells migrating faster than the IgM positive ones. Amongst five patients with chronic lymphocytic leukemia four with IgD positive lymphocytes (greater than 80%) showed an identical electrophoretic distribution. The IgM positive lymphocytes (greater than 80%) of the fifth patient migrated much more slowly. The weighted mean of each distribution profile of either the IgD or IgM positive lymphocytes in CLL is similar to that of normal subjects.     

Preparative free flow electrophoresis for the isolation of two populations of proximal cells from the rabbit kidney

High voltage free flow electrophoresis is a carrier-free method used for analytical and preparative cell separation, based on charge surface properties of cells. Two cell populations from the proximal tubule of the rabbit kidney were isolated by free flow electrophoresis from a suspension of pure proximal cells. This single-cell suspension was obtained through an original method by the combination of a Ca-binder action and gentle mechanical treatment associated with several shifting steps, on a pure suspension of isolated proximal tubules. Before the electrophoretic separation, the proximal cell origin was confirmed by enzymatic marker measurements, and the metabolic capacity was assessed by the cell respiratory activity. The isolated cells were very poor in distal tubule marker enzymes and were enriched in proximal tubule marker enzymes. Respiratory measurement showed a high cell metabolic capacity. After the electrophoretic separation, the origin of the cell populations was assessed by measuring specific marker enzymes. The cells in the slow-moving electrophoresis fractions had a high gamma-glutamyl transpeptidase activity and a low glucose-6-phosphatase activity. The fast moving cells showed a high glucose-6-phosphatase content and a poor gamma-glutamyl transpeptidase activity. Cells isolated by free flow electrophoresis were shown to possess long microvilli. This new methodology, allowed for the first time, the separation of a fast-moving cell population originating from the convoluted portion of the proximal tubule and a slow-moving cell population originating from the straight part of the proximal tubule of the rabbit kidney.     

Separation of human X and Y spermatozoa by free-flow electrophoresis

Free-flow electrophoresis is a fast and promising method for gamete separation. Pretreated seminal plasma-free human spermatozoa were injected continuously as a fine stream into the buffer medium of the separation chamber flowing perpendicular to the forces of an electrical field, which separated the spermatozoa according to their differences in electrophoretic mobility. For characterization of the two classes of spermatozoa before and after separation, quinacrine mustard staining was used to identify the Y-chromosome-bearing spermatozoa carrying the fluorescent body (F-body). Human spermatozoa moved toward the anode and were separated into two main peaks. The faster moving fraction contained nearly 80% Y-bearing spermatozoa and the slower peak consisted mainly of pure X-bearing spermatozoa. Whereas sperm viability as determined by eosin staining was nearly unchanged, sperm motility was reduced after free-flow electrophoresis.