Protein Mobility Shifts Contribute to Gel Electrophoresis Liquid Chromatography Analysis

Profiling of cellular and subcellular proteomes by liquid chromatography with tandem mass spectrometry (MS) after fractionation by SDS-PAGE is referred to as GeLC (gel electrophoresis liquid chromatography)-MS. The GeLC approach decreases complexity within individual MS analyses by size fractionation with SDS-PAGE. SDS-PAGE is considered an excellent fractionation technique for intact proteins because of good resolution for proteins of all sizes, isoelectric points, and hydrophobicities. Additional information derived from the mobility of the intact proteins is available after an SDS-PAGE fractionation, but that information is usually not incorporated into the proteomic analysis. Any chemical or proteolytic modification of a protein that changes the mobility of that protein in the gel can be detected. The ability of SDS-PAGE to resolve proteins with chemical modifications has not been widely utilized within profiling experiments. In this work, we examined the ability of the GeLC-MS approach to help identify proteins that were modified after a small hairpin RNA-dependent knockdown in an experiment using stable isotope labeling by amino acids in cell culture-based quantitation.     

Gel Permeation Chromatography of Polysaccharides: Universal Calibration Curve

Changes in the crystallinity and polymorph of chitosan, which may affect its functionality, by heating (up to 200°C) its water suspension were studied by X-ray diffraction measurements, using tendon chitosan prepared by N-deacetylation of a crab tendon chitin, and chitosan powders with various degrees of polymerization (DPv = 1,720–12,600) and N-acetylation (zero to 26%). It was found that the presence of hydrated polymorphs or anhydrous crystals in a chitosan sample could be examined easily by measuring the powder diffraction pattern of a sample. Chitosan with a low molecular weight or low degree of N-acetylation was highly crystallized, especially in the anhydrous form that is considered to spoil chitosan’s functionality, by heating.     

The Heterogeneity of the 7S Soybean Protein by Sepharose Gel Chromatography and Disc Gel Electrophoresis

Two kinds of N-acetylmuramidase, M-1 and M-2 enzymes, that were isolated from the cultural broth of Stm. globisporus 1829, were remarkably different in amino acid composition, immunological properties and modes of lytic action from each other. The M-1 enzyme was composed of 186 amino acid residues of which two moles were of half cystine, while the M-2 enzyme was composed of 99 amino acid residues with no cysteine. The hydrolyzing action of the M-2 enzyme was suppressed by the presence of an O-acetyl group on muramic acid residues in the peptidoglycan moiety, while that of the M-l enzyme was independent of the presence of O-acetyl groups. However, the hydrolyzing activity of both enzymes was enhanced when some muramic acid residues were substituted with stem peptides containing alanine, isoglutamine and lysine.     

硅胶柱层析法从栀子中分离制备京尼平甙

采用硅胶柱层析法从中药山栀子中分离制备主要药效成分京尼平甙,色谱条件:常规柱(2.5×30cm),固定相:柱层析硅胶;流动相∶乙醇-石油醚(1∶5,1∶3)。样品通过薄层层析以及600~190nm波长扫描定性鉴定后,用高效液相色谱法(HPLC)检测纯度,结果表明纯度为97.6%。     

凝胶层析在发酵液中分离提纯透明质酸中的应用

采用葡聚糖凝胶G-100,以0.1 mol/L氯化钠溶液为洗脱剂,在优化的凝胶层析条件(层析柱高度30 cm、进样量2 ml、洗脱流速1 ml/4 min)下,对透明质酸发酵液进行分离纯化,得到了高纯度的透明质酸.HA提取率达到79.85%,蛋白质去除率为84.93%.     

Dissociation of Oxyhaemoglobin,Methaemoglobin and their Hybrid compared by Difference Gel Chromatography

ACCURATE measurement of the energy of protein subunit interaction^1–7 is an essential step in the study of cooperative and allosteric^8–10 effects in proteins. It is, however, more often the change of this energy on uptake of ligand or substrate which is the more important quantity to be determined, not its total value. Likewise it is the variation of interaction energy from protein to protein which is of more interest when phylogenetically related proteins are compared and again it is differences in energy of combination which determine how much hybrid is formed when closely related reversibly dissociating proteins are present in mixture in solution.     

高效凝胶色谱法测定聚乙烯亚胺的分子量及其分布

目的:探讨高效凝胶色谱法测定聚乙烯亚胺(PEI)及其衍生物的分子量及其分布.方法:采用Ultrahydrogel 250(300mmx7.8mm)色谱柱;以已知相对分子量的PEI为标样;醋酸-醋酸盐缓冲液(0.2mol/l醋酸-0.1mol/l醋酸钠)为流动相;柱温40℃;流速lml/min;示差折光检测器.结果:测得自制PEI衍生物的重均分子量(Mw)为5093,数均分子量(Mn)为2090、Z均分子量(Mz)为11031,分布宽度(Mw/Mn)为2.44.结论:高效凝胶色谱法操作简单、灵敏度高,适合于快速、简便地测定PEI及其衍生聚合物的分子量及其分布.     

Gel Formation in Protein Amyloid Aggregation: A Physical Mechanism for Cytotoxicity

Amyloid fibers are associated with disease but have little chemical reactivity. We investigated the formation and structure of amyloids to identify potential mechanisms for their pathogenic effects. We incubated lysozyme 20 mg/ml at 55C and pH 2.5 in a glycine-HCl buffer and prepared slides on mica substrates for examination by atomic force microscopy. Structures observed early in the aggregation process included monomers, small colloidal aggregates, and amyloid fibers. Amyloid fibers were observed to further self-assemble by two mechanisms. Two or more fibers may merge together laterally to form a single fiber bundle, usually in the form of a helix. Alternatively, fibers may become bound at points where they cross, ultimately forming an apparently irreversible macromolecular network. As the fibers assemble into a continuous network, the colloidal suspension undergoes a transition from a Newtonian fluid into a viscoelastic gel. Addition of salt did not affect fiber formation but inhibits transition of fibers from linear to helical conformation, and accelerates gel formation. Based on our observations, we considered the effects of gel formation on biological transport. Analysis of network geometry indicates that amyloid gels will have negligible effects on diffusion of small molecules, but they prevent movement of colloidal-sized structures. Consequently gel formation within neurons could completely block movement of transport vesicles in neuronal processes. Forced convection of extracellular fluid is essential for the transport of nutrients and metabolic wastes in the brain. Amyloid gel in the extracellular space can essentially halt this convection because of its low permeability. These effects may provide a physical mechanism for the cytotoxicity of chemically inactive amyloid fibers in neurodegenerative disease.     

双向凝胶电泳缺陷胶及其原因

双向凝胶电泳是蛋白质组学研究中的关键技术之一,涉及较多的实验步骤和试剂,常出现缺陷胶而导致实验失败。从数千张电泳胶图中选取了21张典型的缺陷胶图,对它们进行了归类和总结,分析和讨论了形成缺陷胶的多种原因,提出了实验改进的方法和注意事项。     

Studies on the Mechanism of Strengthening Fish Gel by Basic Amino Acids

An electro-energizing fermentation (E-E F) method has been developed. In this method, a direct electrical current is applied to a microbial culture to accelerate the reductive metabolism of microorganisms or to impart profitable effects to microbial cells. This E-E F method was applied to l-glutamic acid fermentation by Brevibacterium flavum No. 2247. When glucose was used as a substrate, the addition of 0.01 mm neutral red (NR), redox dye (electron carrier), to the fermentation broth at the beginning of cultivation was effective for l-glutamate (l-Glu) production. A direct current of 200~300 μA/cm² at 1.5 V was applied through out the cultivation of this bacterium. This resulted in about a 10% increase in yield of l-Glu.     

Purification of the Caenorhabditis elegans Transposase Tc1A Refolded during Gel Filtration Chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12–16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

Analysis of Amylose-borate Interaction by Frontal Gel Filtration

Amylose-borate interaction has been analyzed by frontal gel chromatography, using the constituent velocity data alone. The constituent Velocity equation was reformulated in terms of elution volume for a type of interacting system described by$\text{A}+i\text{B}={\text{AB}}_i(i=1,2,3\dots n)$

Detailed examination of the binding data indicates that, in the complex formation between amylose and borate, this type of equilibria operates predominantly, if not solely. Use of the constituent elution volume equation enabled us, for the first time, to evaluate the association constant (K) and number of binding site pertaining to this system, i.e., K = 4.9 10² and n = 1. There was no evidence indicating the occurrence of the formation of inclusion complex.     

双向电泳关键因素分析

蛋白质组学已成为继基因组学之后生命科学研究的重点,作为其"开门技术"的双向电泳也渐成为人们关注的焦点.本文总结了国内外研究进展,并对影响双向电泳的关键因素进行分析.     

Analysis of Distributed Growth of Saccharomyces cerevisiae Cells Immobilized in Polyacrylamide Gel

A technique is described for the quantitative determination of the distributed growth of Saccharomyces cerevisiae immobilized in polyacrylamide gel. Gel specimens were embedded in paraffin or gelatin and paraffin before sectioning and staining. Photomicrographs of specimen sections were enlarged, and cell microcolony volumes were determined as a function of position in the gel by grid transparency analysis. Overall cell densities within the gel were calculated for a quantitative comparison with values measured by a second spectrophotometric method. The results show good agreement and demonstrate the sigmoidal growth of the immobilized cells, reaching a maximum steady-state value. The technique shows promise as a general method for following the transient growth of organisms immobilized within gel particles.     

Comparative Study between Gas Chromatography and Ion-exchange Chromatography on Amino Acid Analysis of Foods

Lactobacillus brevis and Saccharomyces cerevisiae were completely sterilized by the supercritical (SC) CO2 micro-bubble method. Gaseous (G) and liquid (LQ) CO2 were used in a similar manner to compare the sterilizing effect. Among the three treatments, the microorganisms were only effectively sterilized by the SC CO2 treatment at 25 MPa and 35°C.     

流感病毒的裂解气相色谱分析

裂解气相色谱法(Pyrolysis Gas Chromatography,PGC)在微生物学的应用中曾多侧重于细菌的鉴定,Mayer最早用PGC做植物病毒的快速鉴定,80年代国内开始开展了病毒的PGC研究。本文报道用PGC分析流感病毒和新城疫病毒的初步结果。