Journal of biochemical and biophysical methods最新文献

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.

Myrosinase is a β-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 Brassica carinata, B. oleracea var. capitata, B. napus and Sinapis alba. Myrosinases in the Brassicas formed complexes of different molecular weight (500–600 kDa, 270–350 kDa and 140–200 kDa) whereas in seeds of S. alba 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.

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.

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.

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.

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.

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.

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.

Workers involved in the manufacture of drug substances may be exposed to active pharmaceuticals by inhalation of drug dusts or droplets which has been considered the main exposure route. The proposed HPLC method allowed to determine sulpiryde, hydroxyurea and dyprophylline in the concentration range of 0.01–0.187 mg/m³, 0.001–0.08 mg/m³ and 0.01–0.40 mg/m³ for sulpiryde, hydroxyurea and dyprophylline, respectively, when 480 L of air sample was collected on the glass fibre filters. Sulpiryde was extracted with a solvent system consisting of acetonitrile–phosphate buffer at pH 3 (85:15, v/v), while the best efficiency of extraction for hydroxyurea and dyprophylline was achieved using water. HPLC analysis of sulpiryde with fluorescence detection was more sensitive (LOD = 3.1 μg/L) in comparison with UV detection (LOD = 84.4 μg/L).

The transforming growth factor-β (TGF-β) family is involved in a variety of physiological processes, and transmits signals through phosphorylation of Smad by the receptor complexes. In the present study, effects of blocking solution in Western blot analyses on detection of phosphorylated Smad1/5/8 and Smad2 were examined. When EzBlock was used as a blocking reagent, phosphorylated Smad1/8 and Smad2 were most efficiently detected. The anti-phospho-Smad2 antibody specifically recognized the phosphorylated form of Smad2, whereas the anti-phospho-Smad1/5/8 antibody also reacted to the unphosphorylated form. These antibodies did not react with the other Smads.

Most of the β-blocking drugs for treating diseases of the cardiovascular system are chiral aryloxy–propanolamine derivatives. Tipically, the S(−) enantiomers are more active than the R(+) 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.

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.