Two-dimensional gel electrophoresis of proteins for genetic studies in douglas fir (Pseudotsuga menziesii)

A method of two-dimensional gel electrophoresis of proteins from Douglas fir needles is described. Extraction in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol followed by heating at 100°C produces reliable two-dimensional gels which are convenient for genetic studies. Three genotypes from different geographical origins have been compared: among 225 loci expressed, 22 display regulatory variations and 7 show allelic variations. Thus it is now possible to undertake the genetic study of Douglas fir using this powerful technique.This work was supported in part by Grant ATP PIRDES 508 444 from the INRA-CNRS.     

Use of a prototype gel hardness tester to demonstrate the effect of variable calcium concentration of gel rigidity

Summary Construction and operation of a device to measure gel rigidity is described. A commercially available force gauge capable of recording peak force measurements of up to 200±0.1 g is mounted on an easily constructed device consisting of a frame with a motor which moves a platform supporting a Petri plate up or down at a controlled speed. The force gauge probe pierces the gel in the Petri plate, at which time the peak force is recorded to give an estimate of gel hardness. Using both agar and gelrite, an example of the use of the device is given. Tests on a series of different calcium concentrations in gels of varied strengths demonstrate that the patterns of gel hardness differ. Finally, the rigidities of three typical media (half-strength Litvay, DCR, and Murashige and Skoog) on both gel types are compared.     

Characterization of the filamentous hemagglutin from Bordetella pertussis by gel electrophoresis

Summary A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S, and S₂ (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form lb, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S₂, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1.     

Separation of biomacromolecules by electrofiltration through gel layers

A straightforward method for concomitant separation and isolation of biomacromolecules from a mixture in solution was developed. Three gel layers that comprise a middle separation layer of 10% polyacrylamide gel were constructed. This gel system was formed in an electroconcentration apparatus above a collection chamber surrounded at the bottom by a dialysis membrane. The mixture is applied over the gel layers where biomacromolecules are caused to migrate by electrophoresis through the gel system, where they are separated into discrete bands and electroeluted into the collection chamber without dismantling the apparatus. The isolated biomacromolecules are removed from the chamber in a highly pure and concentrated form ready for further investigations. Cooling can be applied throughout the whole process, and the setup and conditions of run can be modified according to the characteristics of the biomacromolecules to be purified. The components of a mixture containing the glycoprotein ovalbumin and bovine serum albumin monomer, dimer, and tetramer were successfully isolated as concentrated and highly pure fractions with good recoveries ranging from 70 to 89%. Other proteins were successfully isolated under denaturing conditions in the presence of sodium dodecyl sulfate (SDS) or 6 M urea.     

Parallel plate equipment for preparation of uniform gel sheets

A method for the preparation of uniform gel-disks for enzyme and cell immobilisation, as well as for characterisation of gel mechanical stability, is described. The apparatus comprises a stainless steel base unit and glass parallel plates, designed to permit easy and fast production of multiple homogeneous gel sheets of variable thickness.     

单细胞凝胶电泳应用研究进展

单细胞凝胶电泳技术是被广泛使用的检测DNA损伤的技术,其具有灵敏度高、简便、经济、快速等各种优点。在近30年来,该技术广泛被应用于遗传毒理、环境监测、医学诊断等研究及各类实验证实方面。近些年单细胞凝胶电泳与荧光原位杂交等其他技术相结合后更是拓宽了它的应用范围,本文主要介绍了单细胞凝胶电泳的原理及优劣,并重点阐明了其在遗传毒理学、细胞凋亡、环境监测、群体生物学、临床诊断与治疗,生物信息学领域上的应用,并讨论了它的发展前景。     

海藻酸-SiO2杂化凝胶固定化洋葱伯克霍尔德茵脂肪酶的研究

利用四乙氧基硅烷（TEOS）原位水解法将SiO2掺杂于海藻酸（ALG）凝胶中,通过双交联制备出新型ALG—SiO2杂化凝胶以固定化洋葱伯克霍尔德菌脂肪酶。结果表明,固定化酶的最优条件：质量分数为2．0％的ALG、0．2mol／LCaCl2、V（ALG）／V（TEOS）为5、加酶量为1gALG加100mg酶粉、固定化60min、采用直径为0．8mm的针头滴定、真空冷冻干燥。在此条件下,酶蛋白的包埋率可达100％,酶活回收率可达91％。固定化酶的最适pH为8．0,最适作用温度为50℃,重复使用8次后,酶活性仍能保持80％以上。ALG—Si02杂化凝胶的场扫描电镜（FESEM）观察发现凝胶的整体构造仍然是海藻酸凝胶骨架;与ALG凝胶平滑的内部相比较,杂化凝胶仍具有完整的网络结构,但内部更为粗糙,结构更为致密。     

A rapid capillary gel electrophoresis method for the quantitative determination of RuBisCo in spinach

A capillary gel electrophoretic (CGE) method for the quantitative analysis of RuBisCo in spinach leaves was developed. RuBisCo was resolved into large and small subunits in the presence of sodium dodecyl sulphate (SDS) by the CGE procedure which enabled the determination of the molecular weight of each unit accurately; the values so determined were in close agreement with those reported using other methods. Advantages of CGE over SDS-polyacrylamide gel electrophoresis and high-pressure gel filtration include decreased sample preparation and analysis time, superior resolution and greater sensitivity permitting reduced sample size and trace analysis. In addition, CGE provided precise quantification of RuBisCo and was demonstrated to be a viable alternative to other available methods of protein analysis.     

Reaction and diffusion in a gel membrane reactor containing immobilized cells

To investigate the effect of diffusional limitations and heterogeneous cell distribution in a gel-immobilized cell system, a gel membrane reactor has been constructed. The reactor consists essentially of a gel layer with immobilized cells, flanked by two well-mixed chambers. Through one chamber substrate is pumped, and this chamber is the equivalent of the outside of a spherical gel bead. The second closed measuring chamber contains a small quantity of liquid that can equilibrate with the inside surface of the membrane, eventually after a long transient. Analysis of the liquid in this chamber can give direct information on substrate and product concentrations at the gel surface, and is and indication of the situation in the center of a gel bead. The gel membrane reactor appears to be an excellent tool to study diffusion and reaction in a gel-containing immobilized cells. A mathematical model with time- and position-dependent cell concentration and diffusion coefficient is described. Experimental data show the effective diffusion coefficient of glucose in an alginate gel to decrease with yeast cell concentration. Moreover, kinetic parameters could be determined, using the mathematical model. Microscopic analysis confirmed the proliferation of the gel-entrapped microorganisms in the outer layer of the matrix, as predicted by the model. Potentially, this type of reactor has a clear potential to study the physiology of gel-immobilized cells. (c) 1992 John Wiley & Sons, Inc.     

An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes

An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex.     

Effects of bacterial agarase on agarose gel in cell culture

Summary Bacterial agarase, concentrated and purified from culture filtrate of agar-degrading bacteria, has been used to clean cells cultured in soft agarose from gel residues. The enzyme also has been used to liquefy the gel directly in the dishes to facilitate the removal of cells. The surfaces of glioma cells from agarase-treated colonies could not be distinguished in the scanning electron microscope from surfaces of cells which had never been in contact with agarose or agarase. This implies that most agarose residues had been removed, and also that the treatment did not seriously alter the cell surfaces. The influence of the agarase treatment also was tested by comparison of the mitotic index and the incorporation of [³H]thymidine in agarase-treated and untreated cells. No effects of the treatment could be seen in these tests. The work has been supported by the Swedish Cancer Society and the Swedish Natural Science Research Council.     

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.     

Apparatus and method for preparative gel electrophoresis

A new apparatus for preparative gel electrophoresis with continuous elution which includes a miniaturized electrode and elution chamber system is described. The design provides high resolution, high yield, applicability for small and large amounts of peptide material, and easy operation. Furthermore, the apparatus enables a very accurate gel column or gel gradient to be formed. A method for preparative gel electrophoresis in sodium dodecyl sulfate which allows the purification of peptides and proteins without concurrently modifying tryptophane residues or blocking N-terminal α-amino groups is also described.     

An agarose gel resolving a wide range of DNA fragment lengths

To resolve DNA fragments ranging from several kilobases to some tens of base pairs in length, an agarose slab gel of steadily increasing thickness has been designed. During electrophoresis a gradient of decreasing electric-field strength is generated throughout the gel from the cathode end to the anode end. Shorter fragments which migrate further are decelerated, resulting in an increased linearity of the relationship between mobility and molecular weight.     

Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chiral chemical absorption property of a cross-linked poly(N-isopropyl acrylamide-co-sodium acrylate) thermoresponsive smart gel

This investigation leads to the chiral chemical absorption property of a thermoresponsive gel material. d ‐(+)‐α‐phenyl ethyl amine was taken as the chiral chemical. The gel material was synthesized by polymerizing 1:1 mole ratio of N‐isopropyl acrylamide and Na‐acrylate along with methylene bisacrylamide (2 wt.% of total monomer) as a cross‐linker using ammonium persulfate as an initiator and N, N, N′, N′‐tetra‐methyl ethylene diamine as an accelerator. The microporous nature of the gel material is observed by scanning electron microscope as well as by surface analysis. It is a pH as well as thermoresponsive gel. The highest gel swelling is observed at around pH 8.2 at room temperature (30 °C). The gel contains carboxylate (―COO^?) group in this slightly alkaline condition. In the ionic state, the mutual repulsion of the ionic groups helps in swelling the gel. The lower critical solution temperature (LCST) of the gel is observed to be about 38 °C, which is higher than that of poly N‐isopropyl acrylamide itself (32 °C). This corroborates with the theory and other reported results that LCST increases with the incorporation of ionic moiety in the cross‐linked copolymer. The chiral chemical absorption of the gel material was monitored by measuring circular dichroism of the chiral compound in the presence and absence of gel using a circular dichroism spectropolarimeter (J‐810 L) (JASCO International Co., Ltd., Tokyo, Japan). About 18% of d ‐(+)‐α‐phenyl ethyl amine is absorbed from its aqueous solution by 0.01 g of dry gel material (particle size: 106–212 µm) at room temperature. The absorption of the chiral compound is reversible with temperature having a sudden jump at LCST (38 °C) of the gel material. Chirality 24:506–511, 2012. © 2012 Wiley Periodicals, Inc.     

Reversed-phase HPLC separation of proteins on chemically bonded silica gel columns

Reversed-phase high-performance liquid chromatographic (RP-HPLC) separation of proteins on chemically bonded silica gel columns is described. Efficiency of nonporous alkylsilyl bonded silica gel is compared with that of a macroporous gel that has been widely used for the purpose. A comparative study of the separation under conventional and fast separation conditions is also given. The fast separation technique on the nonporous reversed-phase column has the advantage of improving the recovery of late-eluting hydrophobic and large proteins, such as ovalbumin and apoferritin.     

Physical and nutritional factors in gel culture of mammalian cells

Summary The growth of human glioma cells, cultured as spherical colonies in agarose gel, stopped after about 10 days for both large and small colonies apparently due to an increased osmolality in the gel. When osmolality was kept under control by addition of distilled water, growth continued. However, a continuous increase in the population-doubling period, similar for both large and small colonies, then was observed. The increase persisted although excess amounts of nutrition were added. When the cells were cultured in liquid suspension above a thin layer of agarose gel and most of the medium was repeatedly changed, the colonies continued to grow beyond the limits in gel culture. HeLa and hamster embryonic lung cell colonies showed a growth pattern in agarose gel similar to the glioma cells. The results imply that the osmolality must be kept under precise control to prevent growth inhibition. However, it seems difficult to ascertain optimal growth in gel culture for more than about 2 weeks probably because of the accumulation of toxic products. The work was supported financially by the Swedish Cancer Society and the Swedish Natural Science Research Council.     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.