Home About us Contact
|
Home About us Contact | ||
|
|
||
Purification Methods (purification + methods)
Selected AbstractsA Graphene Oxide,Streptavidin Complex for Biorecognition , Towards Affinity PurificationADVANCED FUNCTIONAL MATERIALS, Issue 17 2010Zunfeng Liu Abstract In our postgenomic era, understanding of protein-protein interactions by characterizing the structure of the corresponding protein complex is becoming increasingly important. An important problem is that many protein complexes are only stable for a few minutes. Dissociation will occur when using the typical, time-consuming purification methods such as tandem affinity purification and multiple chromatographic separations. Therefore, there is an urgent need for a quick and efficient protein-complex purification method for 3D structure characterization. The graphene oxide (GO)·streptavidin complex is prepared via a GO·biotin·streptavidin strategy and used for affinity purification. The complex shows a strong biotin recognition capability and an excellent loading capacity. Capturing biotinylated DNA, fluorophores and Au nanoparticles on the GO·streptavidin complexes demonstrates the usefulness of the GO·streptavidin complex as a docking matrix for affinity purification. GO shows a high transparency towards electron beams, making it specifically well suited for direct imaging by electron microscopy. The captured protein complex can be separated via a filtration process or even via on-grid purification and used directly for single-particle analysis via cryo-electron microscopy. Therefore, the purification, sample preparation, and characterization are rolled into one single step. [source] Self-Propagating Domino-like Reactions in Oxidized GraphiteADVANCED FUNCTIONAL MATERIALS, Issue 17 2010Franklin Kim Abstract Graphite oxide (GO) has received extensive interest as a precursor for the bulk production of graphene-based materials. Here, the highly energetic nature of GO, noted from the self-propagating thermal deoxygenating reaction observed in solid state, is explored. Although the resulting graphene product is quite stable against combustion even in a natural gas flame, its thermal stability is significantly reduced when contaminated with potassium salt by-products left from GO synthesis. In particular, the contaminated GO becomes highly flammable. A gentle touch with a hot soldering iron can trigger violent, catastrophic, total combustion of such GO films, which poses a serious fire hazard. This highlights the need for efficient sample purification methods. Typically, purification of GO is hindered by its tendency to gelate as the pH value increases during rinsing. A two-step, acid,acetone washing procedure is found to be effective for suppressing gelation and thus facilitating purification. Salt-induced flammability is alarming for the fire safety of large-scale manufacturing, processing, and storage of GO materials. However, the energy released from the deoxygenation of GO can also be harnessed to drive new reactions for creating graphene-based hybrid materials. Through such domino-like reactions, graphene sheets decorated with metal and metal oxide particles are synthesized using GO as the in situ power source. Enhanced electrochemical capacitance is observed for graphene sheets loaded with RuO2 nanoparticles. [source] Self-Propagating Domino-like Reactions in Oxidized GraphiteADVANCED FUNCTIONAL MATERIALS, Issue 17 2010Franklin Kim Abstract Graphite oxide (GO) has received extensive interest as a precursor for the bulk production of graphene-based materials. Here, the highly energetic nature of GO, noted from the self-propagating thermal deoxygenating reaction observed in solid state, is explored. Although the resulting graphene product is quite stable against combustion even in a natural gas flame, its thermal stability is significantly reduced when contaminated with potassium salt by-products left from GO synthesis. In particular, the contaminated GO becomes highly flammable. A gentle touch with a hot soldering iron can trigger violent, catastrophic, total combustion of such GO films, which poses a serious fire hazard. This highlights the need for efficient sample purification methods. Typically, purification of GO is hindered by its tendency to gelate as the pH value increases during rinsing. A two-step, acid,acetone washing procedure is found to be effective for suppressing gelation and thus facilitating purification. Salt-induced flammability is alarming for the fire safety of large-scale manufacturing, processing, and storage of GO materials. However, the energy released from the deoxygenation of GO can also be harnessed to drive new reactions for creating graphene-based hybrid materials. Through such domino-like reactions, graphene sheets decorated with metal and metal oxide particles are synthesized using GO as the in situ power source. Enhanced electrochemical capacitance is observed for graphene sheets loaded with RuO2 nanoparticles. [source] Charting protein complexes, signaling pathways, and networks in the immune systemIMMUNOLOGICAL REVIEWS, Issue 1 2006Angela Bauch Summary:, Systematic deciphering of protein,protein interactions has the potential to generate comprehensive and instructive signaling networks and to fuel new therapeutic and diagnostic strategies. Here, we describe how recent advances in high-throughput proteomic technologies, involving biochemical purification methods and mass spectrometry analysis, can be applied systematically to the characterization of protein complexes and the computation of molecular networks. The networks obtained form the basis for further functional analyses, such as knockdown by RNA interference, ultimately leading to the identification of nodes that represent candidate targets for pharmacological exploitation. No individual experimental approach can accurately elucidate all critical modulatory components and biological aspects of a signaling network. Such functionally annotated protein,protein interaction networks, however, represent an ideal platform for the integration of additional datasets. By providing links between molecules, they also provide links to all previous observations associated with these molecules, be they of genetic, pharmacological, or other origin. As exemplified here by the analysis of the tumor necrosis factor (TNF)-,/nuclear factor-,B (NF-,B) signaling pathway, the approach is applicable to any mammalian cellular signaling pathway in the immune system. [source] Rapid purification of active ,-secretase, an intramembrane protease implicated in Alzheimer's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Matthias Cacquevel Abstract ,-Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-, precursor protein generates the amyloid-, protein (A,) of Alzheimer's disease, partially inhibiting ,-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of ,-secretase, but at low resolution (15 Å). The limited amount of purified ,-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four ,-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active ,-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme. [source] Evaluation of spore extraction and purification methods for selective recovery of viable Bacillus anthracis sporesLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2001D.C. Dragon Aims: To investigate methods of improving anthrax spore detection with PLET. Methods and Results: Comparisons were made of PLET and blood-supplemented PLET to recover and distinguish spores of a variety of Bacillus species. Heat and ethanol purification of spores, and spore extraction from soil with water and high specific gravity sucrose plus non-ionic detergent, were also carried out. Conclusions: PLET was more selective and suitable than blood-supplemented PLET for detection of anthrax spores in the environmental specimens. However, PLET is not an optimal spore recovery medium. Purification of spores with ethanol was as effective as heat purification. High specific gravity sucrose plus detergent extraction solutions may be more sensitive than extraction with water. Significance and Impact of the Study: This study highlights shortcomings with the standard PLET isolation of anthrax spores and describes ways in which the procedure may be improved. [source] Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracyclinePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2006Changxin Xu Abstract The elucidation of the molecular details of antibiotic resistance will lead to improvements in extending the efficacy of current antimicrobials. In the current study, proteomic methodologies were applied to characterize functional outer membrane proteins (Omps) of E.,coli,K-12 responded to tetracycline and ampicillin resistance for understanding of universal pathways that form barriers for antimicrobial agents. For this purpose, E.,coli,K-12 expressional outer membrane proteome was characterized and identified with the use of 2-DE and MALDI-TOF/MS methods. Then, differential Omps due to tetracycline or ampcilin resistance were determined by comparison between tetracycline minimum inhibitory concentration (MIC)10, ampicillin,MIC10, control0 and control10, showing 9,proteins with 11,spots for tetracycline and 8,protein with 9,spots for ampicillin, showing a difference in only 1,protein (decreased LamB in tetracyclin) between the two antibiotics. Among the proteins, 3,were known as antibiotic-resistant proteins, including TolC, OmpC and YhiU, while FimD precursor, LamB, Tsx, YfiO, OmpW, NlpB were first reported here to be antibiotic-resistance-related proteins. Our findings will be helpful for further understanding of antibiotic-resistant mechanism(s). This study also shows that the combination of Omp purification methods certainly contributes the sensitivity of Omp detection. [source] Survey of albumin purification methods for the analysis of albumin-organic toxicant adducts by liquid chromatography-electrospray ionization-tandem mass spectrometryPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2005Carrie L. Young Abstract HSA has been shown to react with many organic toxicants to form adducts that are useful biomarkers for exposure. Albumin isolation is an important first step for the analysis of these protein-toxicant adducts. We tested several approaches to isolate albumin from serum treated with an electrophilic organic toxicant known to form adducts with albumin, i.e., sulfur mustard agent (HD) (2,2'-dichloroethyl sulfide), in order to evaluate these techniques as purification methods. To select the most efficient isolation strategy, methods were evaluated using gel electrophoresis, total protein quantitation, and peptide-adduct identification by MS. Results suggest that the albumin-rich fractions obtained can be used to identify exposure by quantitating the albumin adducts to electrophilic organic toxicants such as HD. The HiTrap Blue HP albumin isolation system appears to display the most promising results for purifying albumin to detect HD-adducts, exhibiting high purification efficiency, satisfactory albumin recovery, promising specificity, and a higher loading capacity for serum. [source] The spliceosome: the most complex macromolecular machine in the cell?BIOESSAYS, Issue 12 2003Timothy W. Nilsen The primary transcripts, pre-mRNAs, of almost all protein-coding genes in higher eukaryotes contain multiple non-coding intervening sequences, introns, which must be precisely removed to yield translatable mRNAs. The process of intron excision, splicing, takes place in a massive ribonucleoprotein complex known as the spliceosome. Extensive studies, both genetic and biochemical, in a variety of systems have revealed that essential components of the spliceosome include five small RNAs,U1, U2, U4, U5 and U6, each of which functions as a RNA, protein complex called an snRNP (small nuclear ribonucleoprotein). In addition to snRNPs, splicing requires many non-snRNP protein factors, the exact nature and number of which has been unclear. Technical advances, including new affinity purification methods and improved mass spectrometry techniques, coupled with the completion of many genome sequences, have now permitted a number of proteomic analyses of purified spliceosomes. These studies, recently reviewed by Jurica and Moore,1 reveal that the spliceosome is composed of as many as 300 distinct proteins and five RNAs, making it among the most complex macromolecular machines known. BioEssays 25:1147,1149, 2003. © 2003 Wiley Periodicals, Inc. [source] Enzymatic production of ,- D -glucose-1-phosphate from trehaloseBIOTECHNOLOGY JOURNAL, Issue 9 2010Jef Van der Borght Abstract ,- D -Glucose-1-phosphate (,Glc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of ,Glc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60°C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, ,Glc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline ,Glc1P is now available from the cheap substrates trehalose and inorganic phosphate. [source] Allyl-thiosulfinates, the Bacteriostatic Compounds of Garlic against Helicobacter pyloriBIOTECHNOLOGY PROGRESS, Issue 1 2004Pablo Cañizares Allicin and allyl-methyl plus methyl-allyl thiosulfinate from acetonic garlic extracts (AGE) have been isolated by high-performance liquid chromatography. These compounds have shown inhibition of the in vitro growth of Helicobacter pylori(Hp), the bacterium responsible for serious gastric diseases such as ulcers and even gastric cancer. A chromatographic method was optimized and used to isolate these thiosulfinates. The method developed has allowed the isolation of natural thiosulfinates extracted from garlic by organic solvents and is an easy and cheap methodology that avoids complex synthesis and purification procedures. The capacity and effectiveness of isolated natural thiosulfinates have been tested, and this has enabled the identification of the main compounds responsible for the bacteriostatic activity shown by AGE origin of these kinds of organosulfur compounds along with ethanolic garlic extracts (EGE). Additionally, microbiological analyses have suggested that these compounds show a synergic effect on the inhibition of the in vitro growth of Hp. The results described here facilitate the process of obtaining garlic extracts with optimal bacteriostatic properties. The product is obtained in a way that avoids expensive purification methods and will allow the design of live tests with the aim of investigating the potential for the use of these garlic derivatives in the treatment of patients with Hp infections. [source] | ||||||||||