Journal of biochemical and biophysical methods最新文献

{"title":"Fluorescent method for detection of cleaved collagens using O-phthaldialdehyde (OPA)","authors":"Katrina Go,&nbsp;Yousuke Horikawa,&nbsp;Ricardo Garcia,&nbsp;Francisco J. Villarreal","doi":"10.1016/j.jbbm.2007.05.004","DOIUrl":"10.1016/j.jbbm.2007.05.004","url":null,"abstract":"<div><p>Analysis of collagen degradation remains an important but cumbersome task. Traditional methods with dansyl chloride derivatization of collagen have been used to quantify collagen damage. Fluorescent labeling reagents have been developed that offer advantages such as greater solubility in water and low background emission. One such reagent is <em>o</em>-phthalaldehyde (OPA). In this study, we used OPA as a means of detecting small amounts of degraded collagen. Collagen samples isolated from skin or heart were used for OPA conjugation to exposed amino termini (“opalation”). Experiments utilizing small samples aliquoted in microtiter plates were performed to evaluate effects of increasing concentrations of OPA, varying concentrations of collagen, and effects of matrix metalloproteinase (MMP) digestion. Results indicate that within 10 min of reaction, OPA can be used to detect relative differences in cleaved <em>vs.</em> uncleaved collagen from skin or heart. Heart samples obtained from regions of high MMP activity correlated with increased OPA fluorescence relative to tissue with lower MMP activity. On the basis of these results, we conclude that OPA has valuable practical advantages for analytical use in detecting cleaved collagen in small tissue samples.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 878-882"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.05.004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26781388","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A simple spectrophotometric streptavidin–biotin binding assay utilizing biotin-4-fluorescein","authors":"Mark J. Waner,&nbsp;David P. Mascotti","doi":"10.1016/j.jbbm.2007.06.001","DOIUrl":"10.1016/j.jbbm.2007.06.001","url":null,"abstract":"<div><p>A new assay for biotin binding capacity of Streptavidin (SA) is presented in this work. The assay is based on the large decrease in the extinction coefficient at 493 nm that accompanies binding of biotin-4-fluorescein (B4F) to SA. This decrease is attributed to formation of a charge transfer complex between the B4F-donor and one or more SA residues. We show that one may observe the stoichiometric binding via monitoring the absorbance at 493 nm using either SA or B4F as the titrant. The sensitivity of the assay is at the lower end of similar fluorimetric and photometric assays. Though the sensitivity is not substantially lower than other comparable techniques, this assay allows one added flexibility in working range and instrumentation, since the same stock solutions may be used for this new photometric assay or the fluorescence assay for which this ligand was first developed.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 873-877"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.06.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26863046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Filtration performance of microporous ceramic supports","authors":"Aissa Belouatek ,&nbsp;Abdellah Ouagued ,&nbsp;Mustapha Belhakem ,&nbsp;Ahmed Addou","doi":"10.1016/j.jbbm.2007.08.004","DOIUrl":"10.1016/j.jbbm.2007.08.004","url":null,"abstract":"<div><p>The use of inorganic membranes in pollution treatment is actually limited by the cost of such membranes. Advantages of inorganic membranes are their chemical, thermal and pH properties. The purpose of this work was the development of microporous ceramic materials based on clay for liquid waste processing. The supports or ceramic filters having various compositions were prepared and thermally treated at 1100 °C. The results show that, at the temperature studied, porosity varied according to the support composition from 12% for the double-layered (ceramic) support to 47% for the activated carbon- filled support with a mean pore diameter between 0.8 and 1.3 μm, respectively. Volumes of 5 l of distilled water were filtered tangentially for 3 h under an applied pressure of 3.5 and 5.5 bar. The retention of tubular supports prepared was tested with molecules of varying size (Evans blue, NaCl and Sacharose). The study of the liquid filtration and flow through these supports showed that the retention rate depends on support composition and pore diameter, and solute molecular weight. The S1 support (mixture of barbotine and 1% (w/w) activated carbon) gave a flux for distilled water of 68 L/m² h while the double-layered support resulted in a flux of 8 L/m² h for the same solution at the pressure of 3.5 bar. At a pressure of 5.5 bar an increase in the distilled water flux through the various supports was observed. It was significant for the S1 support (230 L/m h).</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1174-1179"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.08.004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41003600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An electrochemical sensor for determination of calcium dobesilate based on PoPD/MWNTs composite film modified glassy carbon electrode","authors":"Xiuhua Zhang,&nbsp;Shimin Wang,&nbsp;Li Jia,&nbsp;Zuxun Xu,&nbsp;Yu Zeng","doi":"10.1016/j.jbbm.2007.10.002","DOIUrl":"10.1016/j.jbbm.2007.10.002","url":null,"abstract":"<div><p>A poly-<em>o</em>-phenylenediamine and multi-wall carbon nanotubes composite (PoPD/MWNTs) modified glassy carbon electrode (GCE) was prepared by in situ electropolymerization using an ionic surfactant as the supporting electrolyte. The morphology of the resulting PoPD/MWNTs composite was characterized by TEM and the electrochemical properties of the modified electrode were characterized by cyclic voltammetry. The electrochemical behavior of calcium dobesilate on PoPD/MWNTs modified electrode was also investigated. The large current response of calcium dobesilate on PoPD/MWNTs modified electrode is probably caused by the synergistic effect of the electrocatalytic property of PoPD and MWNTs. The reductive peak current increased linearly with the concentration of calcium dobesilate in the range of 0.1–1.0 μmol/L and 4.0–400 μmol/L by square wave adsorptive stripping voltammetry, respectively. The detection limit (three times the signal blank/slope) was 0.035 μmol/L. The modified electrode could eliminate the interference of dopamine, norepinephrine and epinephrine at 100-, 90- and 70-fold concentration of 1.0 μmol/L calcium dobesilate, respectively. The proposed modified electrode provides a new promising and alternative way to detect calcium dobesilate.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1203-1209"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.10.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41019681","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proceedings of the 12th International Symposium Of Chemistry, Miercurea Ciuc (Csíkszereda), Romania, October 5-8, 2006; and proceedings of the 7th International Symposium and Summer School on Bioanalysis, Pécs, Hungary, June 10-17, 2007.","authors":"","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"1232-323"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27572293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of chemical structure in stereoselective recognition of β-blockers by cyclodextrins in capillary zone electrophoresis","authors":"László Gagyi ,&nbsp;Árpád Gyéresi ,&nbsp;Ferenc Kilár","doi":"10.1016/j.jbbm.2007.10.004","DOIUrl":"https://doi.org/10.1016/j.jbbm.2007.10.004","url":null,"abstract":"<div><p>Most of the β-blocking drugs for treating diseases of the cardiovascular system are chiral aryloxy–propanolamine derivatives. Tipically, the <em>S</em>(−) enantiomers are more active than the <em>R</em>(+) enantiomers. Only some of them (for example timolol) are used as single enantiomers, the others are employed as racemates. For the determination of the enantiomeric purity of timolol European Pharmacopoeia prescribes an HPLC method using chiral stationary phase. However, the use of chiral capillary zone electrophoresis for the determination of the enantiomeric purity is of pharmaceutical interest. This study describes the application of various cyclodextrin derivatives, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, sulphated β-cyclodextrin and sulphated α-cyclodextrin for the stereoselective analyses of β-blockers. Baseline separation was obtained for bopindolol, carvedilol, mepindolol, pindolol and alprenolol, while only partial separation was observed for sotalol, propranolol, oxprenolol, atenolol, bisoprolol, bupranolol, and metoprolol. The uneven molecular recognition of the enantiomers of the β-blockers, especially of the optical isomers of labetalol and nadolol, showed the importance of the chemical nature of the separators and the analytes.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1268-1275"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.10.004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72221043","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biophysical characterization of double-stranded oligonucleotides using ETBR and isothermal fluorescence spectroscopy: Implication for SNP genotyping","authors":"Pradip Bhattacharya,&nbsp;Swarkar Sharma,&nbsp;Sailesh Gochhait,&nbsp;Rameshwar N.K. Bamezai","doi":"10.1016/j.jprot.2007.10.001","DOIUrl":"10.1016/j.jprot.2007.10.001","url":null,"abstract":"<div><p>The UV-absorption, fluorescence and CD spectra of aps 23 bp oligoduplexes were performed for potential diagnostic purpose. These oligonucleotide sequences were mimicked from natural mutations (mitochondrial genome) of human population (unpublished). This work was designed on the basis of hybridization of non-self complementary oligoduplexes (aps) containing no mismatch, one-mismatch and two-mismatches. Since melting temperature™ is dependent on concentration of the oligoduplex, various concentrations were used in this study protocol. The thermal spectra profiles (UV absorbance and fluorescence) of these oligoduplexes (aps) are different for a particular concentration, and can be implicated for mutations. −<!--> <!-->d<em>F</em>/d<em>T</em> (or d<em>A</em>/d<em>T</em>) vs <em>T</em>, ln<em>K</em> (or <em>R</em>ln<em>K</em>) vs <em>T_M</em>, Δ<em>G</em> vs <em>T_M</em>, Δ<em>S</em> vs <em>T_M</em> and Δ<em>H</em> vs <em>T_M</em> are also variable for those sequences. All these thermodynamic data were calculated from absorbance (at 260 nm) data. On the contrary to the 23 bp oligoduplexes (aps), the PCR products of 97 bp and 256 bp length were genotyped with ETBR (excitation 530 nm, emission 600 nm) fluorimetrically. But our attempts to genotype these PCR sequences with isothermal UV absorbance spectroscopy were unsuccessful. Isothermal UV absorbance spectra has a limitation of sequence length. However, the structural conformation (all B-type) of the oligoduplexes (aps) was determined using CD. The minor discrepancy in CD spectra of these oligoduplexes are not significant for mutational analysis. 97 bp nested PCR product was an amplicon having either GcT or AcC mutation of mitochondria of normal human population, whereas 256 bp PCR product was an amplicon of human BRCA2 gene (NCBI Accession No. <span>AY151039</span><svg><path></path></svg>) of chromosome 13 having either A or G mutation at position −<!--> <!-->26.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1163-1173"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2007.10.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27208485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Importance of probe location for quantitative comparison of signal intensities among genes in microarray analysis","authors":"Rei Kakuhata ,&nbsp;Masahiro Watanabe ,&nbsp;Takenori Yamamoto ,&nbsp;Eriko Obana ,&nbsp;Naoshi Yamazaki ,&nbsp;Masatoshi Kataoka ,&nbsp;Toshihiko Ooie ,&nbsp;Yoshinobu Baba ,&nbsp;Tomoshige Hori ,&nbsp;Yasuo Shinohara","doi":"10.1016/j.jprot.2007.12.005","DOIUrl":"10.1016/j.jprot.2007.12.005","url":null,"abstract":"<div><p>In our previous studies, we demonstrated that expression levels of genes determined by Agilent oligoarray system, code G4130A, could be quantitatively evaluated by spike-in of synthetic full-length mRNAs as standards [Kakuhata R, Watanabe M, Yamamoto T, Akamine R, Yamazaki N, Kataoka M, et al. Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis, J Biochem Biophys Methods 2007;70:755–60]. However, in its successor version (Agilent oligo array system, code G4131F), which was established to enable gene expression analysis over a much wider dynamic range, multiple probes are often utilized for evaluation of expression levels of individual genes; and they showed markedly distinct signal intensities. This result indicates that the observed signal intensities in this new version seemed not to simply reflect the transcript levels of individual genes. To understand the factors influencing the signal intensities of probes, we characterized the properties of the probes used in this new array system and the cRNAs formed during the labeling process. Analysis of cRNAs in the reaction mixture, which were hybridized with the arrays, revealed that the cRNAs were not fully transcribed under the conditions used. For this reason, probes located at the 5′ side of the message were found to give lower signals than those at the 3′ end; and the observed signal intensities were dependent upon the location of probes in the mRNA. Analysis of the correlation between signal intensities and locations on mRNAs for larger numbers of probes also supported the idea that probe location is one of the major determinants of signal intensities of probes in microarray analysis.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 926-931"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2007.12.005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27235963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of an abasic site in RNA with stem-loop DNA beacons: Application to an activity assay for Ricin Toxin A-Chain","authors":"Setu Roday ,&nbsp;Matthew B. Sturm,&nbsp;Dukagjin M. Blakaj,&nbsp;Vern L. Schramm","doi":"10.1016/j.jprot.2007.12.010","DOIUrl":"10.1016/j.jprot.2007.12.010","url":null,"abstract":"<div><p>The catalytic ability of Ricin Toxin A-Chain (RTA) to create an abasic site in a 14-mer stem-tetraloop RNA is exploited for its detection. RTA catalyzes the hydrolysis of the <em>N</em>-glycosidic bond of a specific adenosine in the G<strong><u>A</u></strong>GA tetraloop of stem-loop RNA. Thus, a 14-mer stem-loop RNA substrate containing an intact “G<strong><u>A</u></strong>GA” sequence can be discriminated from the product containing an abasic “G<strong><u>ab</u></strong>GA” sequence by hybridization with a 14-mer DNA stem-loop probe sequence and following the fluorescent response of the heteroduplexes. Three DNA beacon probe designs are described. Beacon 1 probe is a stem-loop structure and has a fluorophore and a quencher covalently linked to the 5′- and 3′-ends. In this format the probe–substrate heteroduplex gives a fluorescent signal while the probe–product one remains quenched. Beacon 2 is a modified version of 1 and incorporates a pyrene deoxynucleoside for recognition of the abasic site. In this format both the substrate and product heteroduplexes give a fluorescent response. Beacon 3 utilizes a design where the fluorophore is on the substrate RNA sequence at its 5′-end while the quencher is on the probe DNA sequence at its 3′-end. In this format the fluorescence of the substrate–probe heteroduplex is quenched while that of the product–probe one is enhanced. The lower limit of detection with beacons is 14 ng/mL of RTA.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 945-953"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2007.12.010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27265962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Methodology to label mixed carbohydrate components by APTS","authors":"Annamária Bui ,&nbsp;Béla Kocsis ,&nbsp;Ferenc Kilár","doi":"10.1016/j.jprot.2008.01.009","DOIUrl":"10.1016/j.jprot.2008.01.009","url":null,"abstract":"<div><p>Labelling of carbohydrates with fluorescent dyes is of importance in the analysis of complex oligo- or polysaccharides. The complexity of the hydrolization mixtures of the oligosaccharides can be studied with separation methods after labelling. However, the procedures might provide unexpected phenomena. The experimental circumstances for labelling carbohydrates with 8-aminopyrene-1,3,6-trisulfonic acid were studied and the labelling efficiency of mono and oligosaccharides present in endotoxins was followed by capillary electrophoresis using LIF detection. Significant differences were observed in the labelling of the different sugar molecules, and the lowest reactivity with the dye was observed in the cases of amino-sugars. The results provide a basis for studies in determining the structures of the core parts and the O-chains of bacterial endotoxins.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1313-1316"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2008.01.009","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27300015","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}