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1.
53-fold purified creatine kinase is isolated from beef heart mitochondria by phosphate buffer extraction followed by chromatography on DEAE-cellulose and KM-cellulose and preparative electrophoresis in phosphate buffer density gradient. The purified enzyme was homogenous under electrophoresis in agarose gel and moved to cathode. The enzyme did not enter into separating gel under disc electrophoresis in conditions for the separation of neutral anc acid proteins, while under conditions for separating alkaline proteins it produced five fractions. The stability of creatine kinase under storage considerably decreased after the purification. 相似文献
2.
Apolipoprotein E was isolated from human very low density lipoproteins by a two-step electrophoretic procedure derived from that of Méndez (1982. Anal. Biochem. 126: 403-408). It included separation in a sodium dodecylsulfate polyacrylamide slab gel, transfer into an agarose gel, and extraction by ultracentrifugation for 30 min. No protein labeling, dialysis, or concentration procedures were needed. The method was fast, showed an excellent protein recovery, and could be suitable as a general method of protein isolation by polyacrylamide gel electrophoresis. 相似文献
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We developed a simple DNA elution method from agarose gels. After electrophoresis of DNA in an agarose gel, the DNA fragment to be recorved was excised out of gel with a scalpel. The excised gel was placed in the middle of small Parafilm piece, and the Parafilm was folded over the gel piece. Using the petriplate, or thumb, the gel piece was pressed between the Parafilm. Upon squeezing, the DNA inside of the gel gets extruded along with the buffer. The droplets were collected with a pipet. The DNA was then purified by conventional phenol: chloroform extraction method. Typical yields are greater than 50% as determined by UV absorbance. 相似文献
5.
A method has been described for the isolation of three differently charged isohormones of rat prolactin from a discard fraction obtained after extraction of gonadotropins, thyrotropin and growth hormone from homogenized frozen pituitaries. The procedure involved extraction at pH 9.8, ammonium sulphate fractionation, molecular sieve chromatography on Sephadex G-100, and column electrophoresis in agarose suspension. The purification was monitored by radioimnunoassays and the recovered components were all found to possess a specific immunoactivity exceeding that of the standard preparation (RP-1) supplied by the NIAMDD, Bethesda, U.S.A. Increased acidity among these isohormones was found to be paralleled by significantly decreased immunopotency. Each component showed biological activity in radioreceptor assay. A high degree of purity of the isolated components was shown by analytical electrophoresis in polyacrylamide gel. Sodium dodecyl sulphate electrophoresis in the same medium showed no size heterogeneity and yielded a value of approximately 25 000 for the molecular weight of the isohormones. In addition a large form of prolactin, suggested to represent a dimer, was isolated by a further extraction step (pH 10.5) followed by molecular sieve chromatography on Sephadex G-100 and electrophoresis in agarose suspension. The large form was associated with both biopotency and immuno-potency. The electrophoresis resolved the prolactin activity into three or four immunoactive components. This pleomorphism of the large prolactin was confirmed by analytical polyacrylamide gel electrophoresis. Amino acid analyses revealed a close similarity between the three monomers and the major dimeric form of the hormone. 相似文献
6.
Pei Yun Lee John Costumbrado Chih-Yuan Hsu Yong Hoon Kim 《Journal of visualized experiments : JoVE》2012,(62)
Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments 相似文献
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目的:通过对TRIzol一步法进行改进,建立一种从富含胶原蛋白、多糖及色素的仿刺参体壁提取总RNA的有效方法。方法:样品在液氮中研磨并用TRIzol匀浆后再进行抽提;对TRIzol一步法提取的总RNA进行DNaseⅠ消化和酚氯仿抽提,用2.5mol/L的醋酸钾沉淀,并加入适量糖原(10mg/mL)与RNA共沉淀。结果:琼脂糖凝胶电泳和紫外分光光度法以及RT-PCR检测结果表明,改进的方法能够有效去除基因组DNA、蛋白、多糖及色素的污染,RNA的产率提高。结论:制备的总RNA纯度高,完整性好,能够满足mRNA差异显示RT-PCR等分子生物学研究的要求,是一种提取仿刺参体壁及其他富含黏多糖、胶原蛋白和色素的动物组织总RNA的有效方法。 相似文献
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E Méndez 《Analytical biochemistry》1982,126(2):403-408
An efficient method for the isolation of a few milligrams of a protein from a protein mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. The method is based on the insertion of an intermediate agarose-containing layer in the polyacrylamide gel. The protein mixture labeled with fluorescamine and the unlabeled one were run simultaneously in separate slots. During electrophoresis the fluorescent-conjugated protein bands were followed by uv illumination. The electrophoresis was stopped when the fluorescent band corresponding to the protein to be isolated was in the agarose layer. The protein is extracted quantitatively from the agarose in less than 1 h by ultracentrifugation. The pure protein recovered in the supernatant was used directly, in the Tris-sodium dodecyl sulfate buffer, to prepare rabbit antiserum. 相似文献
9.
P Upcroft 《Analytical biochemistry》1987,162(1):1-4
Transfection of African green monkey kidney cells directly with recombinant DNA excised from, but still present in, Seaplaque agarose after electrophoresis, is described. Efficiencies of transfection increased by 30% when the gel was present compared with transfection in the absence of the agarose. Extraction of the DNA from the gel was not necessary, thereby obviating a purification step and the concomitant losses. To generate recombinant molecules bacterial plasmid sequences are not necessary, thereby reducing considerably the size of the recombinant molecule and removing extraneous and deleterious sequences, e.g., "poison sequences." Linear or circular DNA molecules could be transfected in the melted and diluted agarose with the same ease as in its absence. Hence linear partial ligation products can be excised from the gel after electrophoresis to generate recombinant DNA molecules directly in mammalian cells. 相似文献
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目的:探讨肿瘤坏死因子α基因外显子3中rs4645843C/T多态对多囊卵巢综合征患者睾酮浓度的影响。方法:应用聚合酶链反应-SNP敏感性分子开关技术检测130例多囊卵巢综合征(PCOS)患者和175例对照组妇女肿瘤坏死因子-α外显子3中rs4645843C/T多态及其分布,比较rs4645843C/T等位基因与PCOS之间的关系,同时采用化学发光法分析测定血清T水平。结果:PCOS及对照组TNF-α基因rs4645843C/T多态分析均显示有CC,CT,TT三种基因型和C,T两种基因。PCOS组中CC,CT,TT三种基因型和C,T两种等位基因频率分别为34.6%、58.5%、6.9%和63.8%,36.2%,对照组中三种基因型和两种等位基因频率分别为43.4%,53.7%,2.9%和70.3%,29.7%,基因型频率和等位基因频率分布差异在两组中均无统计学意义(P=0.108;P=0.093)。在PCOS患者中,各等位基因及基因型频率分布在高雄激素血症组(T≥0.7ng/mL)和非高雄激素血症组(T<0.7ng/mL)差别亦无统计学意义(P=0.867)。方差分析发现CC,CT,TT三种基因型患者之间血清T... 相似文献
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Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing. 相似文献
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A Pellegrini H R Zweifel R von Fellenberg 《The International journal of biochemistry》1983,15(8):1013-1018
A alpha 2-beta 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. The protein moved in agarose gel electrophoresis just above the beta 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. No serological relation was found between the horse alpha 2-beta 1-glycoprotein and human and bovine plasma proteins. 相似文献
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Aims: To compare three methods for DNA extraction from Mycobacterium bovis , Mycobacterium tuberculosis and Mycobacterium avium subsp. avium .
Methods and Results: The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS 6110 in M. bovis and M. tuberculosis , and of a 1700 bp fragment of FR300 region in M. avium avium .
Conclusions: Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex.
Significance and Impact of the Study: The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis. 相似文献
Methods and Results: The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS 6110 in M. bovis and M. tuberculosis , and of a 1700 bp fragment of FR300 region in M. avium avium .
Conclusions: Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex.
Significance and Impact of the Study: The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis. 相似文献
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Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)–agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDS–agarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in the well. Much more insoluble material was observed with curli-proficient strains than with strains that do not make curli. Both highly purified curli and the insoluble material isolated from an SDS–agarose gel could be dissociated into monomers when treated with formic acid. For quantitation, we immobilized samples in SDS–agarose prior to electrophoresis. This avoids losses during the staining of the gel. Our methods provide a rapid and simple fractionation of curli using equipment that is readily available. 相似文献
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Lectin affinity electrophoresis was applied to the separation of charged, fluorescent conjugates of disaccharides. Four fluorescent conjugates were prepared by reductive amination of alpha-D-Man-(1----3)-D-Man, alpha-D-Gal-(1----4)-D-Glc, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc in the presence of 7-amino-1,3-naphthalenedisulfonic acid. These charged fluorescent-disaccharide conjugates all have identical molecular weight and in the absence of conconavalin A lectin failed to separate either by agarose or by polyacrylamide gel electrophoresis. In the presence of either free or immobilized concanavalin A, agarose gel electrophoresis and polyacrylamide gel electrophoresis could separate the fluorescent conjugate of alpha-D-Man-(1----3)-D-Man from that of alpha-D-Gal-(1----4)-D-Gal, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc. 相似文献
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A fluorographic procedure for the detection of [3H]thymidine-labeled deoxyribonucleic acids electrophoresed in agarose gels was developed. 2,5-Diphenyloxazole (PPO) was added to the agarose solution before pouring of the gel for electrophoresis. This procedure did not interfere with the electrophoretic mobility of the DNA molecules. The radioactive detection efficiency was found to be improved over an existing procedure whereby the agarose gel was infused with PPO after electrophoresis with the aid of acetic acid. 相似文献
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Albalat A Gutiérrez J Navarro I 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2005,142(3):347-354
In the present study, we have examined the effects of insulin and glucagon on the lipolysis of rainbow trout (Oncorhynchus mykiss). To this end, adipocytes were isolated from mesenteric fat and incubated in the absence (basal lipolysis) or presence of different concentrations of insulin and glucagon. In addition, to further elucidate the effects of these hormones in vivo on adipocyte lipolysis, both fasting and intraperitoneal glucagon injection experiments were performed. Basal lipolysis, measured as the glycerol released in the adipocyte medium, increased proportionally with cell concentration and incubation time. Cell viability was verified by measuring the release of lactate dehydrogenase (LDH) activity in the medium. Insulin (at doses of 35 and 350 nM) decreased lipolysis in isolated adipocytes of rainbow trout in vitro, while glucagon was clearly lipolytic at concentrations of 10 and 100 nM. Furthermore, hypoinsulinemia induced by fasting, as well as glucagon injection, significantly increased lipolysis in isolated adipocytes approximately 1.5- and 1.4-fold, respectively, when compared with adipocytes from control fish. Our data demonstrate that lipolysis, as measured in isolated adipocytes of rainbow trout, can be regulated by both insulin and glucagon. These results not only indicate that insulin is an important hormone in lipid deposition via its anti-lipolytic effects on rainbow trout adipocytes, but also reveal glucagon as a lipolytic hormone, as shown by both in vitro and in vivo experiments. 相似文献
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AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories. 相似文献
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目的:观察肝素与23种染料之间的相互作用.方法:利用淀粉琼脂糖凝胶电泳及聚丙烯酰胺凝胶电泳,观察染料加肝素后电泳行为的变化.结果:淀粉琼脂糖凝胶电泳中染料加肝素后多数结果是泳速变慢,有一些留在原点,也有前移、后退者.二氯荧光素、荧光红、荧光素、甲酚红、苯胺蓝、溴酚蓝、茜素红S、胭脂红、亮绿、氨基黑10B、丽春红G、曙红、苯胺黑、氯酚红加肝素后泳速变慢.亚甲蓝、灿烂甲酚蓝、甲基紫加肝素后电泳留在原点.刚果红、洋红加肝素后电泳变化不明显.四氯荧光素、氯化硝基四氮唑蓝加肝素后电泳荧光看不清.聚丙烯酰胺凝胶电泳中溴酚蓝加入肝素后泳速明显变慢,随着肝素量增加溴酚兰的泳速加快.与淀粉琼脂糖凝胶电泳结果一致,但它的电泳结果更明显.结论:本次所研究的23种染料,几乎都能与肝素发生相互作用,给它们的进一步深入研究打下基础. 相似文献