A New Apparatus for Preparative Gel Electrophoresis

Syntheses of two carbonyl derivatives of hydantocidin 1, a potent, naturally occurring herbicide, and their herbicidal activity are described. Spiroimidazolidinone 2, the descarbonyl compound at C9, was prepared by employing reductive demethylsulfurization with tri-n-butyltin hydride as the key step. Another derivative, spiroimidazolinone 10, was obtained from α-azidoamide 8 and benzyl isocyanate via the aza-Wittig reaction. 2 had lost almost all herbicidal activity, whereas 10 retained herbicidal activity against such dicotyledonous weeds as ragweed and cocklebur, but lost activity against monocotyledonous weeds. These results imply the possibility that proper modification of the carbonyl group at C7 of the parent compound would afford hydantocidin analogues possessing crop selectivity.     

Isolation of Major Proteins from Boar Sperm Plasma Membranes by Preparative Gel Electrophoresis and Localization of a Major Polypeptide Using Specific Monoclonal Antibodies

A preparative procedure using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is described for isolating major boar sperm plasma membrane polypeptides (PMPs) in soluble form. Proteins were first separated on 6 mm diameter gels using a pH gradient expanded in the acidic region. The second dimension used 6 mm thick, 10 acrylamide gels. Major proteins identified by Coomassie staining were excised and electroeluted. The procedure was applied to the isolation of a group of proteins in the molecular weight range 40K-50K which comprise a major fraction of the total integral membrane protein in these cells (groups 4 and 5^1,2). Yields of electrophoretically pure soluble polypeptides from these groups were between 0.3 mg-0.5 mg from the processing of 16 gels per week. Electroeluted proteins were also used to elicit monoclonal antibodies tc major proteins. Monoclonal antibodies to the major plasma membrane protein referenced as 4.85 were isolatpd and shown to be specific tc this protein by transblotting precedures. This proteir was primarily localized over the anterior portion of the principal segment of ejaculated sperm by indirect FITC fluorescence microscopy.

The ability to isolate 60–100 mg of plasma membranes per week from the cauda epididymides of boars also permitted developing a procedure for the rapid fractionation of large amounts of detergent sclubilizec plasma membranes by isoelectric focusing in flatbecls of Biogel P200. For the first time, individual proteins of sperm surface proteins can be isolated in large enough amounts to begin detailed biochemical characterization, localization, and functional testing.     

Isolation of cu -Antitrypsin in a New Preparative Electrophoresis Apparatus

The construction and operation of an apparatus is described which enables α-antitrypsin to be isolated from human serum by preparative electrophoresis in polyacrylamide gel. The initial pass through the chamber yields a fraction that is predominantly albumin and several α₁-proteins. After removal of albumin by affinity chromatography, a second pass through the chamber separates the individual α₁-proteins. A volume of 50 ml of serum may be accommodated by the chamber, and the recovery of activity in each step is greater than 60%. The entire procedure may be completed in 36 hrs.     

A Novel Gaussian Extrapolation Approach for 2D Gel Electrophoresis Saturated Protein Spots

Analysis of images obtained from two-dimensional gel electrophoresis (2D-GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software available currently has proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this work, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions, using a two-dimensional least-squares fitting based on a generalized Gaussian distribution. Pixel correction in saturated and smeared spots allows more accurate quantification, providing more reliable image analysis results. The method is validated for processing highly exposed 2D-GE images, comparing reconstructed spots with the corresponding non-saturated image, demonstrating that the algorithm enables correct spot quantification.     

小鼠黑质双向凝胶电泳技术的优化

为了优化小鼠黑质双向凝胶电泳(2-DE)技术,比较了不同样品预处理方法、不同胶条和不同上样量对凝胶电泳结果的影响。研究发现,两种样品预处理方法均可顺利升至最高等电聚焦(IEF)电压(10000V)。与Clean-upkit法相比,TAC/丙酮沉淀法预处理后蛋白得率较低,同时丢失了样品中的部分中、小分子蛋白质。pH3-10L胶条中蛋白点主要分布在中间区域;pH3-10NL胶条对中间区域的蛋白点的分离有所改善;pH4-7胶条中蛋白点分布均匀,横条纹较少。80μg上样量的图谱背景干净,但点数最少(411);180μg上样量的图谱蛋白点较多(883),且圆润、清晰,背景干净,分辨率高;300μg上样量的图谱蛋白点虽然最多(904),但背景较深,部分蛋白点融合,横条纹也较多。研究表明,对于小鼠黑质蛋白质,使用Clean-upkit法对样品进行预处理,选取pH4-7的胶条,采用180μg的上样量能获得背景干净、分辨率高的双向电泳图谱,为帕金森病的生物标志物和药物靶点的研究打下了基础。     

Tetrahymena ubiquitin-histone conjugate uH2A. Isolation and structural analysis

The ubiquitin-histone H2A conjugate, uH2A, of the protozoan Tetrahymena pyriformis was isolated by gel chromatography and octadecylsilyl-silica chromatography, from the fractions on the chromatographic purification of histone H2A [Fusauchi, Y. & Iwai, K. (1983) J. Biochem. 93, 1487-1497]. The uH2A showed an amino acid composition corresponding to the sum of an equimolar mixture of two protozoan H2A variants and protozoan free ubiquitin. N- and C-terminal sequencing of the uH2A, by Edman degradation and carboxypeptidase P digestion, showed a branched structure having two N-terminals, those of the H2A and ubiquitin components, and one C-terminal, that of each H2A variant component. Further structural analyses of the uH2A, by tryptic digestion of citraconylated uH2A and of a ubiquitinated BrCN fragment, showed that the ubiquitin C-terminal Gly-Gly is linked to the epsilon-amino group of either Lys-123, 125, or 126 in the H2A sequence, and that the ubiquitin sequence is similar to that of calf thymus but differs at least in the sequence of residues 12-27. The deducted structure was compared with the only known uH2A structure, that of calf thymus, with special reference to the branched site.     

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.     

Preparative Polyacrylamide-Agarose Gel Electrophoresis of Proteins and RNAS by the Use of New Device

Quantitatively reproducible results were obtained by using a new device for preparative gel electrophoresis combined with polyacrylamide-agarose composite gel. When an adequate gel-buffer system was selected according to the procedure described in this paper, proteins and RNA's were well separated and recovered. The new device for preparative gel electrophoresis and the method for preparation of polyacrylamide-agarose composite gel are presented together with the elution profiles of the recovered substances.     

Fractionation of Subunits in Xylanases from Trichoderma viride with a New Simple Preparative Polyacrylamide Gel Electrophoresis Apparatus

Formic acid was identified as the acidic component of antibiotic K-52B by gas-liquid chromatography, whereas amino sugar I hydrochloride was isolated from the acid hydrolysate as the basic sugar component. On the basis of infrared analyses of constituent oligosaccharides, formic acid was assumed to be linked to the hydroxyl group of galactose in oligosaccharide III. From the results of physicochemical properties and ninhydrin degradation, periodate oxidation and peracetylation studies of amino sugar I hydrochloride, C₈H₁₈N₂O₅. 2HC1 was considered to be a new diaminohexose with a substituent group.     

尖吻蝮蛇毒的聚丙烯酰胺凝胶电泳，冬眠特异蛋白的发现

目的 比较冬眠期、冬眠前后、活动期尖吻蝮蛇毒的组分.方法 聚丙烯酰胺凝胶电泳.结果 发现了冬眠期尖吻蝮蛇毒中有一个特异性增强的蛋白质成分,进入冬眠几天后,该成分就可以检测到,出眠几天后及活动期蛇毒中不可见.结论 该成分应该与尖吻蝮冬眠密切相关,暂且称其为"冬眠特异蛋白".     

常规聚丙烯酰胺凝胶电泳快速检测胰蛋白酶抑制剂的方法

ＳＤＳ—ＰＡＧＥ－　明胶电泳是检测蛋白酶抑制剂的常用方法,基本原理为：聚丙烯酰胺凝胶中加入ＳＤＳ和明胶,　　ＳＤＳ使蛋白酶抑制剂发生可逆变性。电泳完毕,用　Ｔｒｉｔｏｎ　Ｘ－　１００恢复凝胶板中蛋白酶抑制剂的活性,凝胶板置于蛋白酶溶液里保温,含蛋白酶抑制剂部位的明胶不被蛋白酶水解而呈现蛋白染色带。ＳＤＳ－ＰＡＧＥ一明胶电泳检测蛋白酶抑制剂时,在同一块凝胶板上可同时检测酸性、碱性及中性的蛋白酶抑制剂,但电泳后处理（包括复性,酶解,漂洗和染色）时间长（约２０ｈ）,而且对于同一样品中分子量相近的蛋白酶…     

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.     

Analysis of Paramecium Macronuclear DNA Using Pulsed Field Gel Electrophoresis

.We have analyzed the macronuclear DNA of Paramecium tetraurelia using orthogonal-field-altemation gel electrophoresis. The mean size of the linear macronuclear DNA molecules is approximately 450 kb. Less than 6% of the macronuclear DNA is larger than 800 kb. Using pulse times of 20, 40, 60 and 90 s we show that the macronuclear fragment containing the A type variable surface antigen gene migrates reproducibly as a 320-kb linear DNA. Over the same pulse times we describe the unusual migration of the ribosomal RNA gene (rDNA) of P. tetraurelia. At pulse times of 20 and 40 s the rDNA migrates at limit mobility (300 and 500 kb, respectively) whereas with 60- and 90-s pulse times, 2 components of rDNA are observed; 1 fraction independent of pulse time migrating at limit mobility, and a 2nd component migrating between 100-kb and 400-kb linear markers. Based upon previous electron micrographic studies of Paramecium rDNA as well as data presented here we conclude that the majority of Paramecium rDNA molecules are a circular DNA form.     

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.     

Preparative Isolation and Purification of Hydroxyanthraquinones from Rheum tanguticum Maxim. on Normal Phase Silica Gel: Using a Flash Master Personal System

Abstract

A flash chromatography method for preparative separation and purification of five hydroxyanthraquinones from the Chinese medicinal herb Rheum tanguticum Maxim. ex Balf. was developed by using Flash Master Personal⁺ systems. The purities of the products, chrysophanol, physcion, emodin, aloe‐emodin, and rhein were all over 90%, determined by high performance liquid chromatography (HPLC). Their structures were ascertained by EI‐MS, ¹H‐ and ¹³C‐NMR data, and by co‐TLC and HPLC with the authentic samples available in our laboratory.     

Preparative Gel Electrophoresis at High Sample Load. The Effect of Some Experimental Variables on Separation Performance

The separation of model protein pairs (hemoglobin/ albumin, trypsin/chymotrypsin, hemoglobin A/hemoglobin F) was studied in an apparatus for preparative gel electrophoresis at loads up to 40 mg/cm ²of the cross-sectional area of the gel bed. Separation was favored by higher ionic strength and by longer migration path. Under the conditions used and within the load range studied, increasing total protein load had no adverse effect but increased voltage gradient, temperature, or gel strength were all unfavorable.     

Studies of Transferrin Polymorphism in Swedish Cattle Using Agarose Gel Electrophoresis

The polymorphic transferrin picture in the sera from 894 Swedish cattle was investigated with an agarose gel electrophoresis technique. The serum transferrin bands in the electrophoresis pattern were first identified by labelling with 59Fe. Six existing phenotypes based on the alleles TfA, TfD and TfE could be detected. The frequencies of transferrin types and transferrin alleles are presented, and it is concluded that there are great differences in the frequencies between the Swedish Red and White and the Swedish Friesian.     

牛乳碱性蛋白二维凝胶电泳条件的优化

为了在二维凝胶电泳(2-DE)中有效分离牛乳中的碱性蛋白,本试验在等电聚焦上样缓冲液中加入异丙醇和甘油,采用杯上样方式,比较了三丁基磷(TBP)和二硫苏糖醇(DTT)两种还原剂对碱性区域蛋白的分离效果。结果发现,等电聚焦上样缓冲液以TBP为还原剂的碱性蛋白分离图谱水平条纹少,优于以DTT为还原剂图谱。表明等电聚焦上样缓冲液以TBP为还原剂,采用杯上样的方法可以改善牛乳中碱性蛋白的分离效果。     

胆管癌胆汁蛋白质样品制备和双向电泳条件的优化

采用合适的裂解液和沉淀方法,提取胆管癌胆汁中的蛋白质,获得分辨率高、重复性好的蛋白质双向电泳图谱.通过不同裂解液和蛋白质沉淀方法提取胆汁蛋白的效果比较,设计了不同的样品制备方法,并且对双向电泳(2-DE)的条件进行优化.结果显示,试验确定了适合胆管癌胆汁的裂解液配方(LSⅣ),丙酮沉淀的蛋白分布完整,沉淀效率相对较高.高伏时、长时间的等电聚焦可以获得高分辨率、重复性好的蛋白质双向电泳图谱.因此,本方法可以应用到胆管癌胆汁蛋白的提取,也可以对其他体液蛋白质样品的制备和双向电泳提供借鉴.