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Preparation Methods (preparation + methods)
Kinds of Preparation Methods Selected AbstractsEFFECTS OF DIFFERENT SAMPLE PREPARATION METHODS ON STABLE CARBON AND OXYGEN ISOTOPE VALUES OF BONE APATITE: A COMPARISON OF TWO TREATMENT PROTOCOLS*ARCHAEOMETRY, Issue 1 2010C. J. YODER Researchers have long debated the appropriateness of stable isotope analysis of bone apatite to reconstruct the diets of ancient animals. The debate has centred, in part, on diagenesis of bone mineral from interaction with the burial environment. A number of acetic acid treatments are used to remove diagenetic carbonates from samples; however, less is known on how different protocols alter stable isotope values. We compare two common acetic acid solution treatments (0.1 M versus 1.0 M-buffered) to examine the effects on carbon and oxygen isotope values and Fourier transform infrared spectroscopy (FTIR) spectra in human bone from different burial contexts. Results indicate that both treatments have a similar effect on isotope values and FTIR spectra in bone apatite. [source] A Comparison of Four Mohs Tissue Preparation Methods Using Porcine SkinDERMATOLOGIC SURGERY, Issue 9 2010FRCPC, WILLIAM LEAR MD OBJECTIVE Mohs surgery relies on high-quality, rapid tissue preparation and processing. This study evaluated four currently performed tissue preparation and processing methods for speed of processing and depth of cut into the tissue block to achieve a complete high-quality section. METHODS The following four methods were tested: cryoEMBEDDER, float, heat sink, and slide. Standardized specimens of porcine skin were used to ensure uniformity. We measured the time required for a technician to flatten, embed, and cut to the first complete section of each specimen. Additionally, we measured the depth in microns required to cut into an embedded specimen to achieve a complete section. RESULTS There were advantages and disadvantages of each method, and our findings suggest that the heat sink and float methods are more time efficient but that the slide and cryoEMBEDDER methods require less cutting into the specimen to obtain a complete section. The cryoEMBEDDER device used in this study was loaned by cryoEMBEDDER (Salt Lake City, Utah). [source] Comparison of cell block preparation methods for nongynecologic ThinPrep specimensDIAGNOSTIC CYTOPATHOLOGY, Issue 10 2007Kelly Nigro M.D. Abstract The purpose of this study was to compare four cell block (CB) methods in the setting of nongynecologic ThinPrep (TP) specimens. 48 CBs were prepared from 12 nongynecologic TP specimens using the following CB methods: (1) Inverted filter sedimentation (IFS); (2) Thrombin method; (3) Albumin method; (4) Simple sedimentation. Each CB was assigned a cellularity score: 0 no cells, 1+ hypocellular, 2+ hypocellular with tissue fragments, 3+ cellular. A score of 2+ or 3+ was given for 11/12 of thrombin, 7/12 IFS, 5/12 albumin, and 2/12 simple sedimentation CBs. Thrombin CBs demonstrated a pale background clot with evenly distributed cells. Albumin CBs had a cracked uneven background. IFS CBs had a clear background, but were technically difficult and cells appeared artifactually crowded. In the setting of nongynecologic TP specimens, the thrombin CB was easily prepared and produced the best CB in regards to cellularity, cell distribution, and background quality. Diagn. Cytopathol. 2007;35:640,643. © 2007 Wiley-Liss, Inc. [source] Comparison of three cytologic preparation methods and immunocytochemistries to distinguish adenocarcinoma cells from reactive mesothelial cells in serous effusionDIAGNOSTIC CYTOPATHOLOGY, Issue 1 2006(I.A.C.), Junko Ueda Ph.D. Abstract We assessed whether a panel of seven antibodies is useful in the differentiation of adenocarcinoma cells (ACCs) from reactive mesothelial cells (RMCs) in effusion samples and to determine optimal specimen preparation conditions for immunocytochemical analysis of effusion samples. Immunocytochemistry (ICC) was performed on three types of effusion preparations from the same effusion specimens: ethanol-fixed smears, ethanol-fixed cell -blocks, and formalin-fixed cell-blocks. Commercially available antibodies MOC-31, Ber-EP4, CA19-9, CEA, EMA, CA125, and HBME-1 were tested on RMCs from four samples of various etiology and 15 samples of adenocarcinoma from various primary sites. Ethanol-fixed smears showed strong immunoreactivity to all antibodies tested. The immunoreactivity of ethanol-fixed and formalin-fixed cell-blocks was significantly lower with all antibodies except CA19-9. Smear preparations are more sensitive than cell-blocks for immunocytochemical study. A panel of antibodies MOC-31, Ber-EP4, CA19-9, and CEA appears to be suitable to distinguish between ACCs and RMCs. Diagn. Cytopathol. 2006;34:6,10. © 2005 Wiley-Liss, Inc. [source] Monolithic media in microfluidic devices for proteomicsELECTROPHORESIS, Issue 18 2006Kyung Won Ro Abstract Considerable effort has been invested in the development of integrated microfluidic devices for fast and highly efficient proteomic studies. Among various fabrication techniques for the preparation of analytical components (separation columns, reactors, extractors, valves, etc.) in integrated microchips, in situ fabrication of monolithic media is receiving increasing attention. This is mainly due to the ease and simplicity of preparation of monolithic media and the availability of various precursors and chemistries. In addition, UV-initiated photopolymerization technique enables the incorporation of multiple analytical components into specified parts of a single microchip using photomasks. This review summarizes preparation methods for monolithic media and their application as microfluidic analytical components in microchips. [source] Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urineELECTROPHORESIS, Issue 14 2005Dan Theodorescu Dr. Abstract We describe the use of capillary eletrophoresis (CE) coupled with mass spectrometry (MS) to identify single polypeptides and patterns of polypeptides specific for prostate cancer (CaP) in human urine. Using improved sample preparation methods that enable enhanced comparability between different samples, we examined samples from 47,patients who underwent prostate biopsy. Of this group, 21,patients had benign pathology and 26 with,CaP, and these were used to define potential biomarkers, which allow discrimination between these two states. In addition, CE-MS data from these 47,urine samples were compared to that of 41,young men (control) without known or suspected clinical CaP to further confirm the polypeptides indicative for CaP. Upon crossvalidation of the same samples, several polypeptides were selected that enabled correct classification of the CaP patients with 92% sensitivity and 96% specificity. We then examined an additional 474,samples from patients with renal disease enrolled in other studies and found that 14 (3%) had polypeptides suggestive of CaP possibly indicating that they harbor clinical CaP. In conclusion, this early pilot study suggests that CE-MS of urine warrants further investigation as a tool that can identify putative biomarkers for CaP. [source] Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigeraINSECT SCIENCE, Issue 6 2008Li-Zhen Chen Abstract Brush border membrane vesicles (BBMV) isolated from insect midguts have been widely used to study Cry1A binding proteins. Sample preparation is important in two-dimensional electrophoresis (2-DE), so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2-DE analysis. [source] Dental trauma and antemortem tooth loss in prehistoric Canary Islanders: prevalence and contributing factorsINTERNATIONAL JOURNAL OF OSTEOARCHAEOLOGY, Issue 2 2007J. R. Lukacs Abstract Differential diagnosis of the aetiology of antemortem tooth loss (AMTL) may yield important insights regarding patterns of behaviour in prehistoric peoples. Variation in the consistency of food due to its toughness and to food preparation methods is a primary factor in AMTL, with dental wear or caries a significant precipitating factor. Nutritional deficiency diseases, dental ablation for aesthetic or ritual reasons, and traumatic injury may also contribute to the frequency of AMTL. Systematic observations of dental pathology were conducted on crania and mandibles at the Museo Arqueologico de Tenerife. Observations of AMTL revealed elevated frequencies and remarkable aspects of tooth crown evulsion. This report documents a 9.0% overall rate of AMTL among the ancient inhabitants of the island of Tenerife in the Canary Archipelago. Sex-specific tooth count rates of AMTL are 9.8% for males and 8.1% for females, and maxillary AMTL rates (10.2%) are higher than mandibular tooth loss rates (7.8%) Dental trauma makes a small but noticeable contribution to tooth loss among the Guanches, especially among males. In several cases of tooth crown evulsion, the dental root was retained in the alveolus, without periapical infection, and alveolar bone was in the initial stages of sequestering the dental root. In Tenerife, antemortem loss of maxillary anterior teeth is consistent with two potential causal factors: (a) accidental falls while traversing volcanic terrain; and (b) interpersonal combat, including traditional wrestling, stick-fighting and ritual combat. Steep-walled valleys (barrancos) and lava fields (malpaís) required agile locomotion and occasional vaulting with the aid of a wooden staff. Accidental falls involving facial injury may have contributed to AMTL. Traditional conflict resolution involved competitive wrestling (lucha canaria), stick-fighting (juego del palo), and ritualised contests involving manual combat. These activities made a small but recognisable impact on anterior dental trauma and tooth loss. Inter-personal behaviours of such intensity leave their mark on skeletal and dental remains, thereby providing insight into the lives and cultural traditions of the ancient Guanches. Copyright © 2006 John Wiley & Sons, Ltd. [source] Aerobic Oxidation of Alcohols under Mild Conditions Catalyzed by Novel Polymer-Incarcerated, Carbon-Stabilized Gold NanoclustersADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 13 2008Céline Lucchesi Abstract Polymer-incarcerated, carbon-stabilized gold nanoclusters were found to be highly active in heterogeneous catalysis for the oxidation of secondary alcohols using molecular oxygen in aqueous medium. After optimization of catalyst preparation methods, highly loaded and highly effective catalysts were obtained, and a broad range of secondary alcohols could be oxidized by using these catalysts under mild conditions. The catalysts could be recovered and reused several times without significant loss of activity. Moreover, kinetics of the oxidation reaction with (±)-1-phenylethanol was investigated. [source] Taurine concentrations in animal feed ingredients; cooking influences taurine contentJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 7-8 2003A. R. Spitze Summary The aim of this study was to determine the taurine content in a variety of animal feeds. There is very little information on the taurine content of ingredients used in home-prepared diets for dogs and cats, and foods fed to wild animals in captivity. This study reports the taurine content of both common and alternative feed ingredients, and compares taurine loss as a result of different methods of food preparation. Foods were selected based on their use in commercial and home-prepared diets. Animal muscle tissue, particularly marine, contained high taurine concentrations. Plant products contained either low or undetectable amounts of taurine. The amount of taurine that remained in a feed ingredient after cooking depended upon the method of food preparation. When an ingredient was constantly surrounded by water during the cooking process, such as in boiling or basting, more taurine was lost. Food preparation methods that minimized water loss, such as baking or frying, had higher rates of taurine retention. [source] A comparison of EDI with solvent-free MALDI and LDI for the analysis of organic pigmentsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2008Ichiro Kudaka Abstract To evaluate the applicability of EDI to material analysis as a new ionization method, a comparison of EDI with solvent-free matrix-assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI) was made for the analysis of organic pigments, e.g. Pigment Yellow 93, Pigment Yellow 180, and Pigment Green 36, as test samples, which are poorly soluble in standard solvents. In EDI, the samples were prepared in two ways: deposition of suspended samples in appropriate solvents and dried on the substrate, and the direct deposition of the powder samples on the substrate. No matrices were used. Both sample preparation methods gave similar mass spectra. Equally strong signals of [M + H]+ and [M , H], ions were observed with some fragment ions for azo pigments in the respective positive or negative mode of operation. For the powder sample of the phthalocyanine pigment PG36, M+, and [M + H]+ in the positive mode and M,, in the negative mode of operation were observed as major ions. Positive-mode, solvent-free MALDI gave M+, [M + H]+ and [M + Na]+ and negative mode gave [M , H], depending on the sample preparation. As solvent-free MALDI, EDI was also found to be an easy-to-operate, versatile method for the samples as received. Copyright © 2008 John Wiley & Sons, Ltd. [source] Characterization of N -palmitoylated human growth hormone by in situ liquid,liquid extraction and MALDI tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2007Emmanuelle Sachon Abstract Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N, group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid,liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N -palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N -palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications. Copyright © 2007 John Wiley & Sons, Ltd. [source] Limitation in obtainable surface roughness of hardened cement paste: ,virtual' topographic experiment based on focussed ion beam nanotomography datasetsJOURNAL OF MICROSCOPY, Issue 2 2008P. TRTIK Summary Surface roughness affects the results of nanomechanical tests. The surface roughness values to be measured on a surface of a porous material are dependent on the properties of the naturally occurring pore space. In order to assess the surface roughness of hardened cement paste (HCP) without the actual influence of the usual sample preparation for nanomechanical testing (i.e. grinding and polishing), focussed ion beam nanotomography datasets were utilized for reconstruction of 3D (nanoscale resolution) surface profiles of hardened cement pastes. ,Virtual topographic experiments' were performed and root mean square surface roughness was then calculated for a large number of such 3D surface profiles. The resulting root mean square (between 115 and 494 nm) is considerably higher than some roughness values (as low as 10 nm) reported in the literature. We suggest that thus-analysed root mean square values provide an estimate of a ,hard' lower limit that can be achieved by ,artefact-free' sample preparation of realistic samples of hardened cement paste. To the best of our knowledge, this ,hard' lower limit was quantified for a porous material based on hydraulic cement for the first time. We suggest that the values of RMS below such a limit may indicate sample preparation artefacts. Consequently, for reliable nanomechanical testing of disordered porous materials, such as hardened cement paste, the preparation methods may require further improvement. [source] A solid-state approach to enable early development compounds: Selection and animal bioavailability studies of an itraconazole amorphous solid dispersionJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2010David Engers Abstract A solid-state approach to enable compounds in preclinical development is used by identifying an amorphous solid dispersion in a simple formulation to increase bioavailability. Itraconazole (ITZ) was chosen as a model crystalline compound displaying poor aqueous solubility and low bioavailability. Solid dispersions were prepared with different polymers (PVP K-12, K29/32, K90; PVP VA S-630; HPMC-P 55; and HPMC-AS HG) at varied concentrations (1:5, 1:2, 2:1, 5:1 by weight) using two preparation methods (evaporation and freeze drying). Physical characterization and stability data were collected to examine recommended storage, handling, and manufacturing conditions. Based on generated data, a 1:2 (w/w) ITZ/HPMC-P dispersion was selected for further characterization, testing, and scale-up. Thermal data and computational analysis suggest that it is a possible solid nanosuspension. The dispersion was successfully scaled using spray drying, with the materials exhibiting similar physical properties as the screening samples. A simple formulation of 1:2 (w/w) ITZ/HPMC-P dispersion in a capsule was compared to crystalline ITZ in a capsule in a dog bioavailability study, with the dispersion being significantly more bioavailable. This study demonstrated the utility of using an amorphous solid form with desirable physical properties to significantly improve bioavailability and provides a viable strategy for evaluating early drug candidates. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3901,3922, 2010 [source] Polymorphism of NCX4016, an NO-releasing derivative of acetylsalicylic acidJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 3 2004A. Foppoli Abstract NCX4016 [2-acetoxybenzoic acid 3,-(nitrooxymethyl)phenyl ester] is a recently developed nitrooxy-derivative of aspirin with improved antiinflammatory, analgesic, and antithrombotic activity as well as increased gastrointestinal safety. Systematic polymorphic screening performed with different solvents and preparation methods resulted in the identification of two polymorphs, designated Forms I and II. They were characterized by scanning electron microscopy, powder X-ray diffraction, thermal analyses, and infrared spectroscopy; the crystal structure of polymorph I was solved by single-crystal X-ray analysis and compared with that of aspirin. Finally, intrinsic dissolution rate studies and calculations according to the melting data method were performed to assess the thermodynamic relationship between the two polymorphs. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:521,531, 2004 [source] Survey of recently board-certified prosthodontists on the board-certification process.JOURNAL OF PROSTHODONTICS, Issue 3 2003Part 2: Preparation, impact Purpose A 2-part survey of recently board-certified prosthodontists was conducted in 2001. The first part of the survey, published in June 2003, determined the trends that assisted the candidates in attaining diplomate status. The second part of the survey was done to determine the preparation methods and resources used to prepare for the examination, the most difficult part of the examination, the most gratifying aspect of becoming board-certified, their current employment status, and whether board certification had any positive impact on their employment. Materials and Methods A questionnaire was mailed to 176 diplomates who had become board certified between the years 1993,2001. Of these, 131 board-certified prosthodontists returned the completed survey, resulting in a response rate of 74%. Results Results from this survey showed that 91% of the diplomates had taken the American College of Prosthodontists (ACP) Board Preparation course; most of the diplomates (41%) prepared for the boards by reviewing prosthodontic literature, reading textbooks, using the ACP Study Guide, and seeking the help of other board-certified prosthodontists; 89% of the diplomates felt that achieving board certification had a positive influence in their employment; 39% of the diplomates indicated that Part 2 of the examination was the most difficult to prepare for, and 41% indicated that Part 2 was the most difficult section; the majority of diplomates (31%) were employed by the military, and the most gratifying aspect of becoming board-certified was personal accomplishment (83%). Conclusions Trends were observed regarding prosthodontists who succeeded with their efforts to challenge the board examination. The majority of the diplomates were employed by the military. Most of the respondents indicated that they took the ACP Board Preparation course and found it helpful. The largest percentage of respondents reported that Part 2 was both the most difficult part to prepare for, as well as the most difficult to complete. The most gratifying aspect of becoming board-certified was personal accomplishment. [source] Structure and Mechanical Properties of Polyurethane/Silica Hybrid CoatingsMACROMOLECULAR MATERIALS & ENGINEERING, Issue 10 2005Yongchun Chen Abstract Summary: Polyurethane/silica hybrid coatings were prepared via in situ (IS) or blending (BL) method using different acidic silica sols. The effects of preparation methods, silica types, and content on the structure and mechanical properties of the hybrid coatings were investigated. It was found that there existed two types of silica phases in the hybrid coatings: silica-rich agglomerate and primary silica-rich phase, whose size and compactness depended upon the silica types and the preparation methods. Introducing silica could result in obvious changes in surface free energy, atomic composition, and mechanical properties of the hybrid coatings. Typical SEM image of the fractured surface of hybrids obtained by the in situ method. [source] Respiratory burst activity of polymorphonuclear cells is dependent on the cell preparation techniqueACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 6 2003J. Zhao Background: Controversial results have been reported regarding the effect of anaesthetics on superoxide anion production during the respiratory burst (RB) of polymorphonuclear cells (PMN). The differences could be caused by the cell preparation methods and the aim of this study was to compare two techniques. Methods: RB activity was measured in cell suspensions isolated with the single-step Ficoll procedure and in unfractionated whole blood. Two concentrations of propofol (therapeutic and 10-fold of this, 6 µg ml,1 or 60 µg ml,1) were investigated after cell preparation with both methods. RB was stimulated with Escherichia coli (E. coli), phorbol 12-myristate 13-acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured by means of fluorescence intensity in a flow cytometer. Results: The percentage of PMNs in whole blood which generate superoxide anions in response to fMLP was significantly lower (2.5 ± 0.7%; mean ± SEM) than that in Ficoll isolated cell suspensions (15.1 ± 1.7%). Incubation with propofol led to a concentration-related decrease of RB activity in Ficoll separated PMNs after both PMA and fMLP stimulation. No significant effect of propofol was observed on the RB in PMA stimulated whole blood samples. Conclusion: The results suggest that the influence of cell preparation methods should be considered when the in vitro effects of anaesthetics on PMN functions are studied with flow cytometric methods. [source] The potential association between fruit intake and body weight , a reviewOBESITY REVIEWS, Issue 6 2009S. Alinia Summary Both national and international bodies recommend an increased intake of fruits and vegetables in order to decrease the risk of overweight and obesity. However, there is a rationale to investigate the separate role of fruits. The aim of this paper was to systematically review and analyse published human intervention, prospective observational and cross-sectional studies on fruit intake and body weight in adults. We identified three intervention, eight prospective observational and five cross-sectional studies that explored this relationship. Two of the intervention studies showed that fruit intake reduced body weight, five of the prospective observational studies showed that fruit consumption reduced the risk of developing overweight and obesity, and four of the cross-sectional studies found an inverse association between fruit intake and body weight. Important methodological differences and limitations in the studies make it difficult to compare results. However, the majority of the evidence points towards a possible inverse association between fruit intake and overweight. Future intervention and prospective observational studies examining the direct and independent role of fruit in body-weight management in free-living individuals are needed. Moreover, important determinants such as energy density, energy content, fruit and vegetable consumption, physical form of fruit and preparation methods need to be included in future studies. [source] Electronic structure of GaN(0001)-2 × 2 thin films grown by PAMBEPHYSICA STATUS SOLIDI - RAPID RESEARCH LETTERS, Issue 5 2008R. Gutt Abstract Gallium nitride thin films were grown on silicon carbide (0001) by plasma-assisted molecular beam epitaxy (PAMBE). The samples were cooled down in nitrogen plasma and characterized in situ by reflection high energy electron diffraction (RHEED), photoelectron spectroscopy (XPS/UPS), and atomic force microscopy (AFM) revealing stoichiometric and smooth GaN films virtually free of contaminations. We present valence band data obtained by UPS with strong emission from surface states inside the fundamental band gap. These states and the observed 2 × 2 surface reconstruction are highly sensitive towards residual molecules. Once these surface states have disappeared the original state could not be recovered by surface preparation methods underlining the necessity of in situ investigations on as-grown surfaces. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert sizePLANT BIOTECHNOLOGY JOURNAL, Issue 3 2004Boulos Chalhoub Summary The construction of bacterial artificial chromosome (BAC) libraries remains relatively complex and laborious, such that any technological improvement is considered to be highly advantageous. In this study, we addressed several aspects that improved the quality and efficiency of cloning of plant genomes into BACs. We set the ,single tube vector' preparation method with no precipitation or gel electrophoresis steps, which resulted in less vector DNA damage and a remarkable two- to threefold higher transformation efficiency compared with other known vector preparation methods. We used a reduced amount of DNA for partial digestion (up to 5 µg), which resulted in less BAC clones with small inserts. We performed electrophoresis in 0.25 × TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer instead of 0.5 × TBE, which resulted in larger and more uniformly sized BAC inserts and, surprisingly, a two- to threefold higher transformation efficiency, probably due to less contamination with borate ions. We adopted a triple size selection that resulted in an increased mean insert size of up to 70 kb and a transformation efficiency comparable with that of double size selection. Overall, the improved protocol presented in this study resulted in a five- to sixfold higher cloning efficiency and larger and more uniformly sized BAC inserts. BAC libraries with the desired mean insert size (up to 200 kb) were constructed from several plant species, including hexaploid wheat. The improved protocol will render the construction of BAC libraries more available in plants and will greatly enhance genome analysis, gene mapping and cloning. [source] The minotaur proteome: Avoiding cross-species identifications deriving from bovine serum in cell culture modelsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2010Jakob Bunkenborg Abstract Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5,15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented. [source] Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2009Anne K. Callesen Abstract Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and rigorous sample collection and sample handling protocols. The sensitivity of the MS analysis relies on the quality of the sample; consequently, the blood sample preparation step is crucial to obtain pure and concentrated samples and enrichment of the proteins and peptides of interest. This review focuses on the serum sample preparation step prior to protein profiling by MALDI MS analysis, with particular focus on various SPE methods. The application of SPE techniques with different chromatographic properties such as RP, ion exchange, or affinity binding to isolate specific subsets of molecules (subproteomes) is advantageous for increasing resolution and sensitivity in the subsequent MS analysis. In addition, several of the SPE sample preparation methods are simple and scalable and have proven easy to automate for higher reproducibility and throughput, which is important in a clinical proteomics setting. [source] Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2009Andreas Richter Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO2 fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO2 flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C4 leaf spiked with C3 starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems. Copyright © 2009 John Wiley & Sons, Ltd. [source] Application of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activitiesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2001Min-Jung Kang Sample preparation methods and data acquisition protocols were optimized for the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to high-throughput quantitative analysis of low molecular mass substrates and products of an enzyme-catalyzed reaction. Using a deuterlum-labeled internal standard, precise standard curves were obtained (r2,=,0.9998) over two orders of magnitude of concentration of rac -1-phenylethylamine (PEA), which is converted to 2-methoxy- N -[(1R)-1-phenylethyl]acetamide (MET) by a lipase-catalyzed reaction with ethylmethoxyacetate (EMA) as second substrate. Reliable relative standard deviations were achieved (,5%) using automated analysis with peak intensity ratios between 0.2 and 5 of analyte to internal standard. This method permitted quantitative analysis of the lipase reaction, producing results comparable to those from gas chromatographic (GC) analysis in the dynamic range of GC. This work shows that MALDI-TOFMS can be applied for the high-throughput screening of enzymes. Copyright © 2001 John Wiley & Sons, Ltd. [source] Maximized PUFA measurements improve insight in changes in fatty acid composition in response to temperatureARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Coby van Dooremalen Abstract A general mechanism underlying the response of ectotherms to environmental changes often involves changes in fatty acid composition. Theory predicts that a decrease in temperature causes an increase in unsaturation of fatty acids, with an important role for long-chain poly-unsaturated fatty acids (PUFAs). However, PUFAs are particularly unstable and susceptible to peroxidation, hence subtle differences in fatty acid composition can be challenging to detect. We determined the fatty acid composition in springtail (Collembola) in response to two temperatures (5°C and 25°C). First, we tested different sample preparation methods to maximize PUFAs. Treatments consisted of different solvents for primary lipid extraction, mixing with antioxidant, flushing with inert gas, and using different temperature exposures during saponification. Especially slow saponification at low temperature (90,min at 70°C) in combination with replacement of headspace air with nitrogen during saponification and methylation maximized PUFAs for GC analysis. Applying these methods to measure thermal responses in fatty acid composition, the data showed that the (maximized) proportion of C20 PUFAs increased at low acclimation temperature. However, C18 PUFAs increased at high acclimation temperature, which is contrary to expectations. Our study illustrates that PUFA levels in lipids may often be underestimated and this may hamper a correct interpretation of differential responses of fatty acid composition. © 2009 Wiley Periodicals, Inc. [source] Self-Assembled Octadecyltrimethoxysilane Monolayers Enabling Selective-Area Atomic Layer Deposition of IridiumCHEMICAL VAPOR DEPOSITION, Issue 7 2006E. Färm A preparation method for octadecyltrimethoxysilane (ODS) self-assembled monolayers (SAMs) that enables the selective-area ALD of iridium is studied using an ALD process for iridium at 225,°C. ODS SAMs were prepared from the gas phase by two preparation methods: either with exposure to ODS only, or with alternate exposures to ODS and water. SAMs were patterned with a lift-off process using aluminium as a mask layer. [source] | ||||||||||||||||||||||