实验8 质粒DNA的提取

一、氯化铯超速离心提取法 1.在LB培养基(10克蛋白胨,5克酵母膏,10克氯化钠,15克琼脂粉,溶解于水后,用1N NaOH调节pH至中性,加水至1,000毫升,高压蒸汽灭菌)上划线接种含有待抽提的质粒的细菌。37℃培养过夜。如果质粒上带有抗性基因,则在培养基内加入相应的抗菌素,例如20微克氨苄青霉素/毫升,12.5—15微克四环素/毫升。 2.挑出单菌落接种在10毫升LB培养液(除不加琼脂粉外,成份与LB培养液相同)中,37℃培养过夜。     

琼脂糖平板制备聚焦电泳

在现有的各种蛋白质、多肽的分析方法中,等电聚焦的高分辨率是十分突出的。但以聚焦电泳来进行制备分离的却并不多,主要原因是缺乏比较实用的方法。我们在对胸腺素组分五做进一步提纯的研究中,建立了琼脂糖平板制备聚焦电泳。据我们的经验,这种方法切实可行,效果良好。一、材料、装置和方法 1、琼脂糖平板的制作将一张面积略大于所要制备的琼脂糖平板的亲水性聚酯膜平放在水平玻璃板上,另外从     

琼脂糖平板制备聚焦电泳

在现有的各种蛋白质、多肽的分析方法中,等电聚焦的高分辨率是十分突出的.但以聚焦电泳来进行制备分离的却并不多,主要原因是缺乏比较实用的方法. 我们在对胸腺素组分五做进一步提纯的研究中,建立了琼脂糖平板制备聚焦电泳.据我们的经验,这种方法切实可行,效果良好. 一、材料、装置和方法 1.琼脂糖平板的制作将一张面积略大于所要制备的琼脂糖平板韵亲水性聚酯膜平放在水平玻璃板上,另外从     

利用放射性~(32)P测定“83-2”杆菌解磷效能的试验

我厂于1971年以磷酸三钙和磷矿粉为磷源,筛选出一株解磷能力较强的细菌,编号“83-2”。经中国科学院微生物研究所初步鉴定为无色杆菌(Achromobacter sp.)。“83-2”杆菌经多次摇瓶测定,其溶磷量:摩洛哥磷矿粉为120微克/毫升;磷酸三钙为600微克/毫升。已     

纸层析法测定庆丰霉素

我们通过试验,找到了一种测定庆丰霉素的纸层析法,此法简易、快速、较准确。现介绍如下。一、试剂层析溶剂:正丁醇:冰乙酸:蒸馏水=2.8:1:1。显色剂:0.3克茚三酮,5毫升吡啶用95%的乙醇定容为100毫升配成。春雷霉素溶液(作定量标准用):贮备液为2毫克/毫升,用时以蒸馏水稀释为500微克/毫升。贮备液可在冰箱中保存三个月。     

TPA促进C_3H/10T1/2细胞转化及有关作用的研究

甲基胆蒽(0.1微克/毫升)不能使C_3H/10T2/1细胞发生转化,但经甲基胆蒽(0.1微克/毫升)启动的细胞,以后用TPA(0.1微克/毫升)连续作用则可以转化。说明TPA具有明显的促进细胞转化作用。TPA不能使经甲基胆蒽启动的细胞的姊妹染色单体交换进一步增加。TPA对C_3H/10T2/1细胞的DNA合成,不论是否先经甲基胆蒽启动,都表现为暂时抑制和以后明显的增强。本文对TPA的促进转化作用及其它有关的生物学作用进行了初步讨论。     

紫貂(Martes zibellina)妊娠期血清孕酮水平的变化

用放射免疫法测定受配雌紫貂妊娠期间(9月至翌年2月)的血清孕酮水平。结果表明:其中产仔雌貂在9月至翌年1 月的血清孕酮呈现相对稳定的较高水平(平均1.45±0.23毫微克/毫升),2月下旬急剧升高至本试验的最高值(平均3.72±1.67毫微克/毫升,P<0.01),空怀雌貂在妊娠前期内的血清孕酮水平均为0.70±0.14毫微克/毫升的低水平,与产仔组相比,差异显著或极显著。作者认为血清孕酮水平的变化与日照和胚泡活动密切相关。     

实验12 cDNA文库的简易构建法

一、合成cDNA单链 1.按照实验3的操作程序,从哺乳类细胞中抽提poly(A)~+mRNA。 2.建立合成cDNA单链的反应系统:50mM Tris·HCI pH8.3(42℃时测定pH值);10mM MgCl_2;10mM DTT;4mM焦磷酸钠;1.25mM dGTP;1.25mM dATP;1.25mM TTP;0.5mM dCTP;15—20μCi α-~32P-dCTP(3000 Ci/mmol);100微克/毫升oligo(dT_12-18);150微克/毫升 Poly(A)~+mRNA;3000单位/毫升反转录酶,用水调节至最终体积为20或40微升。43℃保温30分钟。     

柞蚕蛹抗菌肽D对大肠杆菌K_(12)D_(31)的作用

本文报道了不同浓度的柞蚕蛹抗菌肽D,对大肠杆菌K_(12)D_(31)的杀灭作用动力学。在LEG培养液中,抗菌从D的浓度在5微克/毫升时显效。浓度在10微克/毫升以上时,其杀菌速度大于细菌的增殖速度。固定抗菌肽浓度为10微克/毫升,细菌浓度在3×10~7个细菌/毫升,培养在磷酸钾盐缓冲液中,4小时后能全部杀灭。同样浓度细菌在LEG培养液中,4小时后细菌数下降到约为10~2个细菌/毫升,但不能全部杀灭。同时还提供了柞蚕蛹抗菌肽D和B对大肠杆菌K_(12)D_(31)作用不同时间的电镜照片。     

放射对流免疫电泳自显影术测定血清甲种胎儿蛋白

本文介绍放射对流免疫电泳自显影术测定甲种胎儿蛋白(简称AFP)。此方法具有快速、敏感、简易等特点,是一种比较适合农村的放射免疫测定方法。它的检测范围在25毫微克-25微克/毫升之间, ~(131)I-AFP的有效使用期限较长。用此方法进行普查得到了较为满意的结果。对偶尔发生的非特异性反应,可用放射免疫双向扩散自显影术鉴别。近年来,放射免疫测定血清甲种胎儿蛋白已在临床上广泛应用。饱和分析和放射对流免疫电泳定量法,虽可精确定量10毫微克/毫升左右,但操作过程较长,需要计数仪。为了更好地落实毛主席“把医疗卫生工作的重点放到农村去”的伟大号召,简化放射免疫测定是必要的。标记抗原参人火箭电泳自显影法(简称放射火箭自显影)的建立和应用,为普及放射免疫测定AFP开创了新的途径,但建立多种敏感度高、特异性强、并适合于普查用的放射免疫测定法,仍是临床和研究的需要。本文介绍的放射对流免疫电泳自显影术(简称放射对流自显影),可能成为简单、易行,便于普及的放射免疫测定方法之一。     

Purification of Cytochrome b5 from Pig Testis Microsomes by Isoelectric Focusing in an Immobiline pH Gradient

Testicular cytochrome b5 was purified by a procedure including preparative isoelectrofocusing. The cytochrome b5 was determined to have an isoelectric point of 4.45 on analytical isoelectric focusing. The purified cytochrome b5 was found to be homogeneous and its molecular weight was estimated to be 16,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The oxidized and reduced forms of the purified preparation exhibited absorption spectra of a typical cytochrome b5. A 69-fold purification was achieved with an overall yield of 6.2%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing column chromatography and preparative isoelectric focusing.     

Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing

Testicular cytochrome P-450 was purified by a procedure including preparative isoelectrofocusing. The cytochrome P-450 was determined to have an isoelectric point of 6.47 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited absorption spectrum of a typical cytochrome P-450. 284-fold purification was achieved with an yield of 10.6%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing Aniline-Sepharose 4B column chromatography and preparative isoelectric focusing.     

Two-step purification of cytochrome P-450 from adult pig testis by isoelectric focusing.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing preparative isoelectrofocusing. Purification was achieved 1132 times with a yield of 4.82%. 17alpha-hydroxylase activity was shown to be 14.5 nmol of product/min/nmol of P-450. The cytochrome P-450 was determined to have an isoelectric point of 6.45 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450.     

The purification of rat liver arylhydroxamic acid N,O-acyltransferase

Rat liver arylhydroxamic acid N,O-acyltransferase, a noninducible soluble enzyme that can transform N-hydroxy-N-2-aminofluorenes and N-hydroxy-N-acyl-4-aminobiphenyls into reactive derivatives capable of binding protein and oligonucleotides, has been purified greater than 3000-fold by sequential use of the following methods: homogenization and fractional centrifugation, ammonium sulfate precipitation, chromatography on DEAE-cellulose followed by Sephacryl S-200 filtration, preparative polyacrylamide electrophoresis, and preparative isoelectric focusing. These procedures allowed a 14% recovery of enzyme activity. The molecular weight of the enzyme, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 38,500. The isoelectric point, as determined by preparative and analytical flat-bed isoelectrofocusing, is 4.5; the pH optimum is 7.0. N,O-Acyltransferase showed a Km for N-hydroxy-N-acetyl-2-aminofluorene of 6.3 X 10(-6) M with a Vmax of 10.4 nmol of aminofluorene bound to tRNA/min/mg of protein. Activity was not inhibited by the esterase inhibitor paraoxon. Rat liver N,O-acyltransferase is an enzyme that is very unstable, due in part to labile sulfhydryl groups which easily oxidize in air. The enzyme cannot, however, be fully stabilized with the addition of dithiothreitol.     

Isolation of phosphorylated and dephosphorylated forms of the CP43 internal antenna of photosystem II in Hordeum vulgare L

As a consequence of variation in environmental factors, light being the most important one, a number of photosystem II polypeptides may be reversibly phosphorylated by thylakoid-bound kinase(s). Among them, the reaction centre D1 and D2 polypeptides, the PsbH subunit, and the inner antenna CP43. Here, the separation of two forms of CP43 by high-resolution denaturing polyacrylamide gel electrophoresis is reported. By means of immunoblotting with antibody to phosphothreonine-containing proteins and authentic CP43 and limited proteolysis, these two bands could be identified as the phosphorylated and dephosphorylated forms of CP43. Using non-denaturing isoelectrofocusing, a chromatographically derived CP43-enriched fraction could be resolved into three different native forms of CP43. Among them, one was found to be a phosphorylated form, whereas the other two were dephosphorylated forms of the protein. With respect to other methods, the procedure described here allows the isolation, for the first time, of a fully homogeneous population of this chlorophyll-protein complex, opening the way to the study of the role of phopshorylation on functional properties of this core antenna protein.     

人白血病细胞癌性促凝物(CP)的分离纯化及其特性的初步研究

用三步纯化法从人M_3型白血病细胞中分离纯化出人类肿瘤癌性促凝物(CP)。促凝活性回收率为24％,CP纯化倍数为2481倍。纯化CP在SDS-PAGE上为单一区带,其理化和酶学特性类似于动物肿瘤CP,分子量约为70 000,PI为4.8,在FVⅡ缺乏血浆中以及在含有组织因子(TF)抑制剂情况下仍能激活FX。CP促凝活性能被半胱氨酸蛋白酶抑制剂HgCl_2抑制,纯化CP能与抗动物肿瘤CP抗体形成免疫沉淀反应。     

Detection and isolation of glycoproteins and nuclear proteins in the bovine brain

Using ammonium sulphate precipitation, ion exchange chromatography and preparative isoelectrofocusing, 9 organ-specific glycoproteins and 16 specific nuclear proteins were isolated from bovine brain nervous tissue in a homogeneous state. The isolation of proteins was controlled by a solid phase immunoenzymatic analysis. The molecular weight, subunit composition and isoelectric points of the proteins were determined and their ability to interact with immobilized calf thymus DNA and concanavalin A was demonstrated. It was assumed that the multiplicity of specific proteins of brain tissue is a molecular basis which provides for the functional specificity of the nervous tissue at large.     

Purification and characterization of a colony stimulating factor from human lung.

Conditioned medium prepared from human autopsy lung tissue contains high level activity of colony stimulating factor which stimulates granulocytes and macrophage colony formation in both mouse and human bone marrow. The lung colony stimulating factor has been purified about 2250-fold by methods including hydroxylapatite chromatography, preparative gel electrophoresis, preparative isoelectric focusing, and gel filtration chromatography. The final specific activity was 2.7 X 10(6) units/mg. The purified factor has a molecular weight of 41 000 as determined by gel filtration. It is stable at the pH range of 6.5--10 and 56 degrees C for 30 min but sensitive to protease digestion and periodate oxidation. On polyacrylamide gel electrophoresis, it migrates in the alpha-globulin post-albumin region. Upon isoelectrofocusing lung colony stimulating factor appears heterogeneous with isoelectric points of 3.7--4.3. Treatment with neuraminidase did not affect its activity, but caused a change in electrophoretic mobility and isoelectric point. Antibody produced by immunizing rabbits with partially purified lung colony stimulating factor exerted strong inhibitory activity on the factor from lung as well as on colony stimulating factor from other human sources including serum, urine, and placenta.     

Purification and characterization of cardiac tropomyosins.

A new procedure was developed to purify tropomyosin. The procedure was an adaptation of that described for purification of myosin. By eliminating troponin before precipitating with (NH4)2 SO4, it was possible to obtain pure tropomyosin from the same preparation from which myosin was purified. When tropomyosin was subjected to isoelectrofocusing two tropomyosins were present, having similar isoelectric points of pH 5.4 and 5.6; two tropomyosin subunits were resolved in the presence of 6 M urea. The two subunits had very similar isoelectric points, pH 4.7 and 5.0. According to Ouchterlony analyses the tropomyosins from canine skeletal and cardiac tissue were immunologically identical when incubated with goat gammaG antitropomyosin (cardiac).     

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the and subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75–5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.