Journal of biochemical and biophysical methods最新文献_第7页

Improving parameter estimation for cell surface FRAP data 改进细胞表面FRAP数据的参数估计

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2007-07-19 DOI: 10.1016/j.jbbm.2007.07.002

Omer Dushek, Daniel Coombs

{"title":"Improving parameter estimation for cell surface FRAP data","authors":"Omer Dushek, Daniel Coombs","doi":"10.1016/j.jbbm.2007.07.002","DOIUrl":"10.1016/j.jbbm.2007.07.002","url":null,"abstract":"<div><p>Fluorescence Recovery After Photobleaching (FRAP) using the confocal laser scanning microscope has become a standard method used to determine the diffusion coefficient and mobile fraction of cell surface proteins. A common experimental approach is to bleach a stripe on the cell surface and fit the ensuing FRAP curve to a 1D diffusion model. This model is derived from the time course of recovery to an infinitely long stripe bleached on an infinite flat plane. This choice of model dictates the use of a long bleach stripe. We demonstrate that, in the case of a long bleach stripe, the finite extent of the cell leads to significant errors in parameter estimation. We further show that these errors are reduced when a relatively small stripe is bleached. Unfortunately, diffusion to such a region is fundamentally two dimensional and therefore applying the 1D model of diffusion leads to significant errors. We derive an equation suitable for fitting to FRAP data acquired from small bleach regions and analyze its accuracy using simulated data. We propose that the use of a small bleach region along with a two dimensional diffusion model is the ideal protocol for cell surface FRAP.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1224-1231"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.07.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26895809","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}

引用次数: 17

A fast and gentle method for the isolation of myrosinase complexes from Brassicaceous seeds 一种快速温和的从十字花科种子中分离芥子酶复合物的方法

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2007-11-21 DOI: 10.1016/j.jprot.2007.11.006

Natalia Bellostas, Iben Lykke Petersen, Jens Christian Sørensen, Hilmer Sørensen

{"title":"A fast and gentle method for the isolation of myrosinase complexes from Brassicaceous seeds","authors":"Natalia Bellostas, Iben Lykke Petersen, Jens Christian Sørensen, Hilmer Sørensen","doi":"10.1016/j.jprot.2007.11.006","DOIUrl":"10.1016/j.jprot.2007.11.006","url":null,"abstract":"<div><p>Myrosinase is a <em>β</em>-thioglucosidase glucohydrolase that catalyses the hydrolysis of the thioglucoside bond in glucosinolates, allelochemicals present in Brassicaceous plants. These isoenzymes have been found to form complexes with other proteins; however, traditional isolation procedures involving ammonium sulphate precipitation and/or ion exchange chromatography do not allow for the isolation of these complexes. The present paper reports a fast and gentle procedure for the isolation of myrosinases in the complex form. Partial purification by Con A affinity chromatography followed by Sephadex G-200 gel filtration allowed for the isolation of myrosinase complexes from seeds of <em>Brassica carinata</em>, <em>B. oleracea</em> var. capitata, <em>B. napus</em> and <em>Sinapis alba</em>. Myrosinases in the <em>Brassicas</em> formed complexes of different molecular weight (500–600 kDa, 270–350 kDa and 140–200 kDa) whereas in seeds of <em>S. alba</em> it was only possible to isolate and detect 140–200 kDa complexes. In all species the complexes were formed by isoenzymes with isoelectric points between 4.8 and 5.6 and in some cases up to 6.8. SDS-PAGE confirmed that the myrosinase isoenzymes were composed by several protein subunits of molecular weights ranging between 10 and 110 kDa. The relative amount and enzymatic activity of the myrosinase complexes varied amongst the species studied. The isolation of myrosinase complexes in their native form is of great importance for the study of the hydrolysis of glucosinolates under autolysis conditions.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 918-925"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2007.11.006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27193559","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}

引用次数: 29

A rapid and non leaky way for preparation of the sharp intracellular recording microelectrodes 一种快速无漏制备细胞内记录微电极的方法

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2007-05-29 DOI: 10.1016/j.jbbm.2007.05.007

Masoud Fereidoni , Yaghoub Fathollahi , Mahyar Janahmadi , Iran Godarzi

{"title":"A rapid and non leaky way for preparation of the sharp intracellular recording microelectrodes","authors":"Masoud Fereidoni , Yaghoub Fathollahi , Mahyar Janahmadi , Iran Godarzi","doi":"10.1016/j.jbbm.2007.05.007","DOIUrl":"10.1016/j.jbbm.2007.05.007","url":null,"abstract":"<div><p>To fill microelectrodes using backfilling method needs excessive time approximately 4–6 h. It is often difficult to fill microelectrodes without damage or leakage. A main problem is bubble formation in microelectrodes which has an impact on the electrical properties of the electrode and thus it influences the quality of the recording. Based on Archimede's principle there is a force within a solution which pushes insoluble material with a lower specific gravity upward and outside of the solution. Centrifugation can increase the force to eliminate the bubbles.</p><p>We designed a microelectrode holder to protect microelectrode sensitive tips from mechanical damage due to the gravity tensions; it can help to eliminate the bubbles easily and simultaneously in 10 min or less.</p><p>The tests were performed for 2000, 4000, and 8000 rpm centrifugation each one for 3, 6 and 12 min duration respectively, it was found that the bubbles were completely eliminated at 8000 rpm for 6–12 min and there were no significant differences for resistance, and the number of leaky or damaged electrodes between the two methods.</p><p>In the new design of devices, the materials used and the design of the holder are simple and the approach is applicable to many laboratories worldwide.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1124-1129"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.05.007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26792052","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}

引用次数: 0

Development of potentiometric urea biosensor based on urease immobilized in PVA–PAA composite matrix for estimation of blood urea nitrogen (BUN) PVA-PAA复合基质脲酶固定化尿素电位生物传感器的研制

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2008-01-07 DOI: 10.1016/j.jprot.2007.12.006

Sandeep Kumar Jha , Anita Topkar , Stanislaus F. D'Souza

{"title":"Development of potentiometric urea biosensor based on urease immobilized in PVA–PAA composite matrix for estimation of blood urea nitrogen (BUN)","authors":"Sandeep Kumar Jha , Anita Topkar , Stanislaus F. D'Souza","doi":"10.1016/j.jprot.2007.12.006","DOIUrl":"10.1016/j.jprot.2007.12.006","url":null,"abstract":"<div><p>A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1–1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinity™ BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, β-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1145-1150"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2007.12.006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27311932","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}

引用次数: 45

Optimization of minuscule samples for use with cDNA microarrays 用于cDNA微阵列的微小样品的优化

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2007-12-23 DOI: 10.1016/j.jprot.2007.11.011

Susan McLean Hunter , Fiona C. Mansergh , Martin J. Evans

{"title":"Optimization of minuscule samples for use with cDNA microarrays","authors":"Susan McLean Hunter , Fiona C. Mansergh , Martin J. Evans","doi":"10.1016/j.jprot.2007.11.011","DOIUrl":"10.1016/j.jprot.2007.11.011","url":null,"abstract":"<div><p>The recent advent of microarray technology and RNA amplification allows us to compare the expression profiles of thousands of genes from small amounts of tissue or cells. We have compared and contrasted various methods of RNA preparation, RNA amplification, target labelling and array analysis in order to achieve a streamlined protocol for microarraying small samples. We have concluded that usage of the NIA 15K cDNA array set, in combination with RNA extraction using the Mini RNA Isolation kit (Zymo), amplification with the RiboAmp kit (Arcturus), followed by indirect labelling via the Atlas™ PowerScript™ Fluorescent Labelling kit (using a modified protocol), is optimal with a material derived from either very early stage mouse embryos or individually picked embryonic stem cell colonies. Normalisation using the analysis package Limma (Bioconductor) with data normalisation by print tip Loess, using the “normexp” function with an offset of 50 for background adjustment, and incorporating A-quantile between array normalisation was best with our results. Furthermore, RT-PCR confirmation of array results is achievable without amplification, thereby controlling for amplification bias. These methods will be of great utility in mapping the transcriptome of embryonic and other small samples.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1048-1058"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2007.11.011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27253975","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}

引用次数: 12

Quantification of some active compounds in air samples at pharmaceutical workplaces by HPLC 高效液相色谱法定量制药厂空气样品中某些活性化合物

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2007-10-13 DOI: 10.1016/j.jbbm.2007.10.003

A. Osytek , M. Biesaga , K. Pyrzynska, M. Szewczyńska

引用次数: 7

A sensitive detection of phospho-Smad1/5/8 and Smad2 in Western blot analyses Western blot检测phospho-Smad1/5/8和Smad2的敏感性

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2007-10-10 DOI: 10.1016/j.jbbm.2007.10.001

Masayuki Funaba , Masaru Murakami

引用次数: 22

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. 第12届国际化学研讨会论文集，Miercurea Ciuc (Csíkszereda)，罗马尼亚，2006年10月5-8日;2007年6月10-17日，第7届国际生物分析研讨会暨暑期学校会议记录。

Journal of biochemical and biophysical methods Pub Date : 2008-04-24

引用次数: 0

Cyanine dyes for the detection of double stranded DNA 用于检测双链DNA的花菁染料

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2008-01-07 DOI: 10.1016/j.jprot.2007.12.008

H. Hilal, J.A. Taylor

引用次数: 27

Fluorescent method for detection of cleaved collagens using O-phthaldialdehyde (OPA) 邻苯二醛(OPA)荧光法检测断裂胶原

Journal of biochemical and biophysical methods Pub Date : 2008-04-24 Epub Date: 2007-05-24 DOI: 10.1016/j.jbbm.2007.05.004

Katrina Go, Yousuke Horikawa, Ricardo Garcia, Francisco J. Villarreal

{"title":"Fluorescent method for detection of cleaved collagens using O-phthaldialdehyde (OPA)","authors":"Katrina Go, Yousuke Horikawa, Ricardo Garcia, 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}

引用次数: 23