A Simple Procedure for the Isolation of Brain Actin

Actin has been isolated from bovine brain by forming an actin-containing gel from acetone powders. Solubilization of the gel and chromatography on Sephadex G150 produces actin in good yield. Acetone powders of crude pellets of brain homogenates obtained in the isolation of tubulin produces about 80% of the actin isolated from acetone powder of whole brain. The procedure therefore can be used to isolate both proteins from the same brain.     

Purifying Pararosanilin for Use in Colorless Schiff Reagent

Pararosanilin hydrochloride or pararosanilin base was purified by suspending 20 g. of the dye in 400 ml. of water, acidifying with 50 ml. of 2N HCl and adding 4—5 g. of decolorizing charcoal. The mixture was then heated to boiling and boiled for 2 minutes. The entire mixture was transferred to a large, covered, fluted filter, and the filtrate allowed to stand overnight while the pararosanilin hydrochloride precipitated. The pararosanilin hydrochloride was filtered off, resuspended in 100 ml. of ether-alcohol (10:1) and shaken for 3 to 5 min. The ether-alcohol suspension was filtered and the pararosanilin hydrochloride washed repeatedly on the filter with ether until the ether wash was no longer colored. It was then washed several times with distilled water (total volume 400 ml.), dried in vacuuo over concentrated sulfuric acid, ground to a fine powder and stored in a dark brown bottle.     

制备用于结晶的蛋白质样品

制备高质量蛋白质晶体是通过X射线衍射解析蛋白质分子三维结构的关键环节,是结构生物学领域中的瓶颈问题之一。蛋白质的结晶受多因素控制,其中蛋白质样品自身的质量是影响蛋白质能否结晶及晶体质量好坏的关键因素。我们从蛋白质纯度、可溶性、均一性及表面修饰等方面介绍了如何获得适于结晶的蛋白质样品,以及如何借助相关仪器检测蛋白质样品的质量,预测蛋白质的可结晶性。     

体外诊断试剂防腐剂的选择策略

对体外诊断试剂中防腐剂的作用机理及选择原则进行了阐述,并对目前常用的防腐剂如叠氮钠、庆大霉素、硫柳汞、异噻唑啉酮类等的性能进行了比较,为体外诊断试剂的研究开发及生产中防腐剂的使用提供有益指导。     

SOn-column Protein Refolding for Crystallization

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.     

一种制备酵母菌线粒体DNA的简便方法

ASimpleProcedureforPreparationmtDNAinYeastJinJianlingGaoDongSunZhongdong(MicrobiologyDepartment,ShandongUniversity,Jinan250100)目前,制备酵母mtDNA的常用方法大致有两类：一是先提取混合DNA（核DNA＋mtDNA）,再通过CsCI密度梯度离心或柱层析法分离纯化mtDNA（’,’,”’;二是先通过蔗糖不连续梯度超离心法分离纯化线粒体,再从线粒体中提取mtDNA”’。这些方法虽然能够得到纯度较高的mtDNA,但超离心法需要配套设备（超速离心机等）,柱层析法对样品的回收浓缩比较复杂。本文报道的制备mtDNA的方法,不需…     

A Simple Solid-Phase Based Purification Procedure for Oligodeoxynucleotides

Abstract

A two-cartridge method for routine purification of DNA oligomers has been investigated. The full-length target oligonucleotides are purified using a method that select for intact 3′- and 5′-termini. The procedure results in purified DNA without the use of PAGE gels or HPLC.     

Simplified Manufacture and Histochemical Use of the Schiff Reagent

Schiff reagents were made by two methods. The first procedure gave a Schiff reagent of pH 1.8-2.4. It was accomplished by passing sulfur dioxide into 0.5% aqueous fuchsin solution at room temperature, stopping at reddish violet, and decolorizing allowed to occur on standing. In another method, 1.5 ml. of 5.6% sulfurous acid was added to 100 ml. 0.5% fuchsin solution and the mixture produced in several hours a colorless Schiff reagent of pH 3. The solution remained unchanged for some weeks when kept stoppered in a refrigerator.

To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges. These experiments showed that the Feulgen reaction was optimum at pH 3, the McManus reaction at pH 2.4.     

A Simple Assay Procedure for Mixtures of Hematoxylin and Hematein

A simple and rapid method for the simultaneous quantitative analysis of mixtures of hematoxylin and hematein uses the molar extinction coefficients of the pure substances calculated by Lalor and Martin (1959). Absorbance measurements of the samples dissolved in methanol are made at wavelengths of 292 nm and 445 nm, the wavelengths of maximum absorption of hematoxylin and hematein respectively. The hematoxylin absorbance at 292 nm is corrected for the presence of hematein.

Using this method it was found that of 12 commercial samples labelled “hematoxylin” all contained at least 90% of the compound. Hematein contents of these samples fell in the range 0.1% to 6.8%. In 9 commercial samples labelled “hematein” the hematein contents fell in the range 1.2% to 90.7%. The hematoxylin contents of these samples fell in the range of 1.0% to 82.7%.

This paper describes also a chromatographic method for the identification of hematein and its oxidation products.     

Schiff and pseudo-Schiff reagents: the reactions and reagents of Hugo Schiff,including a classification of various kinds of histochemical reagents used to detect aldehydes

During the 1860’s, Hugo Schiff studied many reactions between amines and aldehydes, some of which have been used in histochemistry, at times without credit to Schiff. Much controversy has surrounded the chemical structures and reaction mechanisms of the compounds involved, but modern analytical techniques have clarified the picture. I review these reactions here. I used molecular modeling software to investigate dyes that contain primary amines representing eight chemical families. All dyes were known to perform satisfactorily for detecting aldehydes in tissue sections. The models verified the correct chemical structures at various points in their reactions and also determined how decolorization occurred in those with “leuco” forms. Decolorization in the presence of sulfurous acid can occur by either adduction or reduction depending on the dye. The final condensation product with aldehyde was determined to be either a C-sulfonic acid adduct on the carbonyl carbon atom or an aminal at the same atom. Based on the various outcomes, I have placed the dyes and their reactions into five categories. Because Hugo Schiff studied the reactions between aldehydes and amines with and without various acids or alcohol, it is only proper to call each of them Schiff reactions that used various types of Schiff reagents.     

Shotgun Crystallization Strategy for Structural Genomics II: Crystallization Conditions that Produce High Resolution Structures for T. maritima Proteins

Currently, 119 high resolution structures of Thermotoga maritima proteins have been determined by the Joint Center for Structural Genomics (JCSG, www.jcsg.org). Sixty-seven of these were solved using the first implementation of the multi-tiered crystallization strategy at the JCSG for the efficient crystallization of large numbers of protein targets. Previously, we reported the analysis of all proteins crystallized using this multi-tiered strategy [Lesley, S.A. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 11664–11669; Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037]. Here, we extend the analysis and describe the crystallization characteristics of those proteins that produced diffraction quality crystals, ultimately resulting in high resolution structures. First, we found that over 77% (52) of the crystals used for structure determination were produced directly from high-throughput coarse screens, indicating that less than one quarter of the crystals (15) required fine screening. In addition, as observed for the proteome screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037], the majority of conditions that produced crystals for natively expressed proteins, whose structures have been determined, were distinct from those of their more extensively purified and selenomethionine-labeled counterparts. Finally, 99% of the proteins whose structures were solved crystallized in conditions contained in the JCSG Minimal Core Screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037; Page, R. and Stevens, R.C. (2004) Methods 34, 373–389], a set of 67 conditions previously identified as those most likely to produce crystals of a diverse set of proteins, confirming its success for rapid identification of proteins with a natural propensity to crystallize.     

电场中的蛋白质结晶

人们一直致力于寻求提高蛋白质晶体质量的方法,利用电场诱导蛋白质结晶即是诸多方法中的一种。已有文献报道显示,电场对蛋白质结晶的影响是积极的。我们从直流电场、交流电场、内置电场、外置电场对蛋白质结晶的影响及相关结晶设备,电场中生长的蛋白质晶体质量的评估,电场中蛋白质结晶的原理及影响因素等方面,对已报道的电场中的蛋白质结晶研究工作进行了总结。     

Crystallization of Synthetic Escherichia coli-Type Lipid A

Synthetic Escherichia coli-type lipid A formed hexagonal plate crystals when it was precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl₂ and incubated in 70% ethanol containing 250 mM MgCl₂ at 4 C for 10 days. Analyses of crystals by electron diffraction and synchrotron X-ray diffraction showed that crystals consist of hexagonal lattices with the lattice constant (a side of the lozenge as a unit cell on the basal plane) of 4.62 Å and the longitudinal axis (perpendicular to the basal plane) of 49.3 ±1.3 Å. Results suggest that the previous finding that various kinds of R-form lipopolysaccharides crystallized but free lipid A isolated by acid hydrolysis from Re lipopolysaccharide did not crystallize under the same experimental conditions (Kato et al, J. Bacteriol., 172: 1516-1528, 1990) is due to structural changes of lipid A occurring during the procedure of isolation of free lipid A.     

A Simple Procedure for Extracting Methacrylic Acid from Water-Miscible Methacrylates

A simple procedure for removing methacrylic acid from water-miscible methacrylates is described. One volume of monomer is diluted with 9 volumes of diethyl ether. Three consecutive extractions are carried out with 5% aqueous NaHCO₃ at 4 C. Residual water is removed by shaking with anhydrous sodium sulfate. The ether is removed by flash evaporation on a Biichi Rotavapor. Weak alkali extraction produces good quality semithin sections which are free of background staining. This method may be a useful alternative to existing methods for removal of methacrylic acid.     

The Recognition of Lipolytic Activity with the Microscope

In view of its probable wide applicability, it seems desirable to publish a note on a simple technic for the recognition with the microscope of the action of lipase. In brief, the method is to make an emulsion of neutral fat previously stained with a red Sudan stain, subject some of the emulsion to the action of the supposed lipase for an appropriate time and then examine with the microscope a recovered drop of the emulsion in a solution of Nile blue sulfate. It has long been known that Nile blue sulfate stains liquid neutral fats a reddish color and fatty acids blue.     

Simple, Inexpensive Procedure for the Disruption of Bacteria

Small volumes (1 to 2 ml) of bacterial cultures, with turbidities ranging from 3 to 10, were disrupted 50 to 90% (measured as a decrease in turbidity) within 2 min, by shaking them on a Vortex-type mixer in the presence of glass beads. This method of disrupton was effective for cells in the following genera: Streptococcus, Lactobacillus, Staphylococcus, Bacillus, and Escherichia. After low-speed centrifugation, the resulting extract can be used as potential sources for enzymes, transforming deoxyribonucleic acids, cell walls, membranes, etc.     

Basic Fuchsin in Acid Alcohol: A Simplified Alternative to Schiff Reagent

The use of Schiff reagent to demonstrate polysaccharides (after prior periodic oxidation) and nucleic acids (after prior acid hydrolysis) is unnecessary since the same results are obtained by substituting a 20 min staining in a 0.5% w/v solution of basic fuchsin in acid alcohol (ethanol-water-concentrated HC1, 80:20:1) followed by a rinse in alcohol. The shade of the basic fuchsin staining is a little yellower than that achieved with Schiff reagent but the selectivity, light fastness, response to different fixatives, and to prior histo-chemical blocking of the tissue section were much the same for the two methods. The need for prior oxidation or hydrolysis and the inhibitory effect of aldehyde blocking techniques indicate that basic fuchsin, like Schiff reagent, reacts with aldehyde groups. Infrared studies indicate that for cellulose the reaction product is an azomethine.     

ToRCH系列酶标诊断试剂的研制

报道了用辣根过氧化物酶标记的抗人ＩｇＧ和抗人ＩｇＭ（ｕ链）单克隆抗体作第二抗体,用自己培养、纯化的弓形体（Ｔｏ）、风疹病毒（ＲｕＶ）、巨细胞病毒（ＣＭＶ）和单纯疱疹病毒（ＨＳＶ１２）的虫体和病毒抗原包被酶标板,研制出检测ＴｏＲＣＨ系列的特异性ＩｇＧ和ＩｇＭ的间接ＥＬＩＳＡ试剂。质量检定结果表明,该试剂特异性强、本底低,能有效消除ＲＦ因子等干扰因素的影响;灵敏度达１∶１６０～６４０;精密性好,变异系数（Ｃ．Ｖ）在１．４％～９．０％;试剂稳定,３７℃存放４ｄ,各项指标的变化率不超过１５％。