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1.
The purification of a hydroxysteroid sulfotransferase from the cytosolic fraction of the hamster epididymis is described using ammonium sulfate precipitation, gel filtration and PAP agarose affinity chromatography. A purification of 360-fold was achieved and resulted in the isolation of one major protein as evidenced by HPLC and SDS gel-electrophoresis. The "native" enzyme is a dimer of mol. wt 106,000 and is composed of subunits having the same molecular weight.  相似文献   

2.
New thiophilic matrices and new procedures were used for the purification of immunoglobulins both from human serum and from hybridoma cell cultures containing fetal calf serum. A range of aromatic and heteroaromatic ligands containing hydroxyl or amino groups have been coupled to divinyl sulfone-activated agarose. The resulting affinity matrices have the general formula M-O-CH2-CH2-SO2-CH2-CH2-X-Y, where M is the agarose matrix, X is oxygen or nitrogen, and Y is an aromatic or heteroaromatic compound. Contrary to earlier expectations these matrices showed pronounced thiophilic binding patterns when tested for the selective binding of immunoglobulins from human serum. The binding is influenced by the structure of the aromatic part of the ligand, the ligand concentration, and the concentration and type of lyotropic salt. 2-Hydroxypyridine coupled to divinyl sulfone-activated agarose was used to purify murine monoclonal antibodies (IgG1 and IgM) from hybridoma cell cultures containing fetal calf serum. Compared to previous methods, significantly increased binding capacity (300-1500%) was obtained by using 1.0-1.2 M ammonium sulfate. Purity of the monoclonal antibody may be optimized for each individual clone by washing the column with either a low concentration of ammonium sulfate or polyethylene glycol before elution.  相似文献   

3.
We found that Toyopearl HW-65C gel matrix adsorbed ferredoxin and ferredoxin-NADP+ reductase in the presence of concentrated ammonium sulfate. Ferredoxin was strongly adsorbed on the gel in 80% saturated ammonium sulfate, and ferredoxin-NADP+ reductase was adsorbed in 40% saturated ammonium sulfate. The phenomenon was utilized for purification of ferredoxin and the reductase on a Toyopearl HW-65C: ammonium sulfate column. The technique greatly simplified the early stage of purification of ferredoxin and the reductase. The improved purification methods further involved column treatments with DEAE-Toyopearl 650M and Matrex Red A. The effectiveness of the columns is reported. Since a number of other proteins such as cytochrome c, myoglobin, chymotrypsinogen A, ovalbumin, and glucose oxidase were also adsorbed well in an appropriately concentrated ammonium sulfate solution, the method may be of general use in enzyme purification.  相似文献   

4.
We have developed a simple, rapid method for purification of beta-glucuronidase from human liver in order to facilitate the study of its biochemical structure and pathophysiologic roles in both cholelithiasis and carcinogenesis. The procedure includes the following steps: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation, and (4) immunoaffinity chromatography employing murine anti-human beta-glucuronidase monoclonal IgG binding to tresyl-activated agarose. beta-Glucuronidase constitutes 1.3 mg per 100 g of wet liver tissue. The enzyme can be purified with a 10% overall yield and overall purification of 5000-fold in a 2-day cycle on a fairly large scale by the method described. Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by protein blotting.  相似文献   

5.
The distribution of proteins on SDS-urea polyacrylamide (7.5%) disc gel electrophoresis is studied from rat brain tubulin purified by three different procedures, including ammonium sulfate precipitation followed by DEAE cellulose chromotography, three cycles of polymerization-depolymerization and colchicinecontaining agarose affinity columns. Three tubulin-associated proteins other than the principal tubulin dimer are identified and characterized with respect to molecular weight, behavior on gel filtration chromatography and method of tubulin purification. One of these proteins (H1) is released from the tubulin complex when colchicine is irreversibly bound to tubulin. These proteins may participate in processes related to microtubule assembly or function.  相似文献   

6.
1. A new purification procedure was started with salting-out fractionation of serum proteins at 45-75% saturated ammonium sulfate concentration, followed by HE agarose affinity chromatography by which calcium-dependently bound CRP and SAP were purely eluted with EDTA-containing buffer. 2. Pure CRP and SAP were finally separated by DEAE-5PW HPLC. 3. This procedure gave recovery of 15 and 26%, and fold purification of 2650 and 2400 for CRP and SAP, respectively. 4. Each subunit of CRP and SAP had one intrasubunit disulfide bond, determined by reduction and carboxymethylation.  相似文献   

7.
8.
M Ugozzoli  A Chiu 《BioTechniques》1992,12(2):187-8, 190
Several components of the extracellular matrix in the molecular weight range of 220 kDa to 150 kDa were purified by preparative electrophoresis on 2.5% Pro-Sieve agarose gels. These high molecular weight glycoproteins, separated under reducing conditions, were recovered in solution by extraction of individual agarose gel slices and analyzed on sodium dodecyl sulfate polyacrylamide gels and Western blots. This simple method permitted the separation and recovery of the laminin B chains (220 kDa and 205 kDa) and entactin (150 kDa) and may prove useful for the purification of other high molecular weight species.  相似文献   

9.
Several prophylactic human papillomavirus (HPV) vaccines have been developed based on virus-like particles (VLPs) made from viral L1 proteins. A substantial number of VLPs is necessary for biochemical characterization and diagnostic test development. To establish the optimum conditions for production and purification of HPV L1 in the yeast expression system we varied the amount and nature of the carbon source and evaluated HPV 16 L1 recovery by three purification methods. Maximally threefold more HPV 16 L1 was produced with a 4% carbon source than with a 2% carbon source. In addition, the productivity of HPV 16 L1 varied by 25% depending on the combination of glucose and galactose in the 4% carbon source. We introduced an ammonium sulfate precipitation step in place of the ultracentrifugation using a sucrose cushion routinely used for HPV L1 purification, and optimized the purification by cation-exchange chromatography. Overall L1 protein recovery using the ammonium sulfate precipitation method was 30%, the highest recovery achieved so far. The purified HPV 16 L1 protein successfully self-assembled into VLPs. Purification by ammonium sulfate precipitation was maximally 15 times greater than ultracentrifugation on a sucrose cushion. We anticipate that our procedures for production and purification will reduce the cost, time and labor involved in obtaining sufficient yields of VLPs.  相似文献   

10.
目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。  相似文献   

11.
This new method allows an easy and rapid purification of amplifiable Escherichia coli plasmids such as pBR 322 without the use of cesium chloride centrifugation. After gentle lysis, centrifugation, and phenol extraction, the material is reextracted with acid phenol to remove the bacterial DNA. The high-molecular-weight ribosomal RNA is removed by precipitation with 2 m ammonium sulfate and the tRNA by passage through a small column of Sepharose CL 4B in the presence of 2 m ammonium sulfate.  相似文献   

12.
We intend to purify beta-glucuronidase from human liver in a large quantity in order to facilitate the study of its biochemical structure and pathophysiologic roles in cholelithiasis and carcinogenesis. The initial purification procedure involved: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation of protein at 56 degrees C, (4) gel filtration with Bio-Gel P-300 gel, (5) anion exchange chromatography with DEAE agarose, (6) cation exchange chromatography with CM agarose, and (7) hydroxyapatite chromatography (overall yield, 1%; overall purification, 169X). The final product was used to immunize rabbits and BALB/c mice for production of polyclonal and monoclonal antibodies, respectively. The antibodies, mainly IgG, were purified by using gamma-Protein A agarose column chromatography. The purified IgG, after periodate oxidation, was coupled to hydrazide gel by formation of a stable covalent hydrazone bond linkage. The new purification procedure involved the initial first three steps, followed by (4) polyclonal IgG immunoaffinity chromatography and (5) monoclonal IgG immunoaffinity chromatography (overall yield, 6.1%; overall purification, 3720X). Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by electroelution. It is concluded that beta-glucuronidase constitutes 0.016 mg per gram of wet liver tissue and can be obtained on a large scale in a highly purified form within a 2-day cycle.  相似文献   

13.
This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.  相似文献   

14.
人尿液中蛋白含量低,在进行质谱分析时易被高丰度蛋白掩盖。因此,发展高效和高选择性的富集方法,是实现尿蛋白标记物深度覆盖的必要前提。探究不同实验方法对尿液蛋白富集和尿蛋白质组的影响尤为重要。本研究采用超滤法、硝酸纤维素膜富集法和饱和硫酸铵沉淀法,等体积各处理5例健康志愿者和膀胱癌患者10 mL尿液样本,富集尿液蛋白,SDS-PAGE分离尿蛋白,比较不同方法纯化的效率;通过质谱分析,比较不同纯化方法的肽段鉴定效果,确定针对尿液蛋白质组蛋白的最佳富集方法。相对于超滤和硝酸纤维素膜富集法,饱和硫酸铵沉淀法成功地应用于健康人尿蛋白的富集和质谱检测,在保证回收蛋白质量的前提下,可减少高丰度白蛋白的干扰,富集更多低丰度蛋白,提高了质谱鉴定的灵敏度。综上所述,饱和硫酸铵提取尿蛋白的效果较好,该方法具有大规模处理尿液、提高蛋白质组学筛选临床诊断标记物研究的应用潜力。  相似文献   

15.
The selective removal of impurity proteins and colloidal particles from milk prior to chromatographic purification processes presents a crucial issue in the production of therapeutic proteins from transgenic animals with high recovery yield and purity. We have developed an efficient two-step precipitation method for the recovery of the recombinant human erythropoietin (rhEPO) of interest from transgenic sow milk. Here, rhEPO was partially purified from transgenic sow milk via a two-step precipitation method consisting of ammonium sulfate and divalent metal precipitations, with a yield of approximately 82.1% and a purification fold of 10.4 at a copper concentration of 30 mM. Copper proved to be the strongest flocculating agent among the divalent ions tested for the aggregation of milk proteins under 35%, with ammonium sulfate, zinc, nickel, and calcium demonstrating increasing flocculating capability in the given order. Copper and zinc proved to be appropriate divalent metals for the recovery of rhEPO at high yield and purity, and the optimal concentration ranges of copper and zinc were 20~40 and 40~80 mM, respectively.  相似文献   

16.
A rapid method for the isolation and purification of small quantities of highly active fatty acid synthetase (FAS) from several strains of the yeast Saccharomyces cerevisiae, is presented. The purification procedure which is the shortest reported to this date (18 hr), involves the release of the enzyme by either cell wall digestion with Zymolyase 60000 or cell wall disruption by glass beads, followed by 35-50% ammonium sulfate fractionation, desalting by Sephadex G-25 chromatography, then calcium phosphate gel treatment, concentration by 50% ammonium sulfate precipitation, sedimentation of the enzyme in the ultracentrifuge and finally, column chromatography on DEAE Bio-Gel A. Fatty acid synthetase prepared by the cell breakage method, was found to be homogeneous according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-Tris-glycine disc gel electrophoresis and immunoelectrophoresis criteria. However, enzyme prepared from Zymolyase treated cells showed several proteolytic bands in addition to FAS bands, on SDS-PAGE. Enzyme obtained by both methods of cell breakage, showed a similar behavior throughout the purification procedure and gave a similar yield of enzyme of high specific activity (4800-7200 nmol/min/mg) that remained stable for several months at -85 degrees C.  相似文献   

17.
A new strategy for protein purification using a soluble affinity matrix is described. The method was used for purification of estrogen receptor. Cytosols from rat uteri and human fibroid uterine tissue, after fractionation by ammonium sulfate, were treated with estradiol-polylysine conjugate. The highly basic affinity complex was separated from other proteins by DEAE-Sephacel chromatography. After dissociation of the eluted complex with excess estradiol, the receptor was recovered by CM-Sephadex chromatography. A 2000-fold purification of the rat uterine estrogen receptor was obtained with an activity recovery of 35%.  相似文献   

18.
《Insect Biochemistry》1987,17(8):1187-1192
Two methods for the purification of vitellogenin from Drosophila melanogaster are described. The first method is a bulk purification starting with approximately 500 g of adult flies reared in population cages. Dimethylformamide and ammonium sulfate fractionations are the bases of this method, and the final yields of purified vitellogenin are on the order of 30 mg. The second method starts with approximately 25 g of adults. Vitellogenin is first enriched by ammonium sulfate fractionation, and then purified by affinity chromatography using rabbit anti-yolk protein (anti-vitellin). Final yields are on the order of 150–200 μg of highly purified vitellogenin. Finally, we show that this purified vitellogenin may be phosphorylated in vitro with the catalytic subunit of protein kinase.  相似文献   

19.
An improved procedure for the isolation of glia maturation factor   总被引:1,自引:0,他引:1  
A procedure for the bulk isolation of glia maturation factor (GMF) in high yield and high purity from bovine brains is outlined. The method involves extraction by homogenization and centrifugation, followed by ammonium sulfate precipitation and column chromatography with diethylaminoethyl (DEAE) Sephacel, Sephadex G-75, and hydroxylapatite. The method results in a 10,000-fold purification, a purity exceeding that of previously published procedures, and enables us to handle as much as 2.8 kg brain tissue or eight brains/week. The ability to mass-produce GMF with this method greatly facilitates its biological studies, further purification, and chemical characterization. The isolated GMF shows a molecular weight of 13,000 on Bio-gel P-30 column and an isoelectric point of about 5.4 on isoelectric focusing. The isolated GMF is heat labile and susceptible to papain and ficin but relatively resistant to trypsin, neuraminidase, and endoglycosidase.  相似文献   

20.
A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.  相似文献   

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