Of this suspension, 25 μl was used to assay for total glutathione

Of this suspension, 25 μl was used to assay for total glutathione (reduced glutathione buy KU55933 + oxidised glutathione ratio – GSH + GSSG) content, while the other 25 μl was treated with

4-vinylpyridine 0.5 μmol/l, a scavenger of GSH, to assay the GSSG content. One hundred twenty-five microlitres of reaction buffer (PBS 143 mmol/l containing 6.3 mmol/l EDTA at pH 7.4, 229 U/ml GSH reductase, 2.39 mmol/l β-nicotinamide adenine dinucleotide phosphate (NADPH) and 0.01 mol/l 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB)) was added to each 25-μl suspension. The conversion of DTNB to 5’-thiol-2-nitrobenzoic acid (TNB) by the oxidation of GSH to GSSG was monitored by measuring absorbance at 405 nm every min over 10 min using a Tecan GENios plate reader. The rate of conversion, measured by the slope of the curve, was proportional to the concentration of glutathione in the sample. A standard curve with different concentrations of GSSG was used

to calculate the glutathione contents in the samples. Statistical analysis For all the assays used, we performed three independent Mdm2 inhibitor experiments with exposures carried out in triplicate for each concentration. The values shown are expressed as mean ± standard error of the mean (SEM). Sigma Plot 12 software (Systat Software Inc, CA, USA) was used for statistical analysis. The normality of the distribution was checked by means of the Shapiro-Wilk test. Equal variance was not assumed by the software and was tested (F test). A one-way repeated measures analysis of GSK923295 research buy variance (RM-ANOVA) was carried out, followed by a post hoc Dunnett’s test with P < 0.05 or P < 0.01. Results Physico-chemical characterisation of PBH-capped AuNPs The AuNPs were synthesised using PBHs as capping ligands (Figure 1). In a previous study [9], we used PBHs containing cysteine (Cys), tyrosine (Tyr) and glycine (Gly) Edoxaban amino acids to form stable AuNPs: Au[(TrCys)2B] and [(Gly-Tyr-TrCys)2B]. In the present study, we demonstrate that the amino acids methionine (Met) and tryptophan (Trp) are also useful to prepare stable functionalised

AuNPs such as Au[(Met)2B], Au[(Gly-Tyr-Met)2B] and Au[(Gly-Trp-Met)2B]. TEM images of the PBH-capped AuNPs and the corresponding size distribution histograms are shown in Figure 2. The micrographs show isolated near-spherical NPs with diameters of 1.5, 1.6, 2.3, 1.8 and 2.3 nm for Au[(Gly-Tyr-Met)2B], Au[(Gly-Trp-Met)2B], Au[(Met)2B], Au[(Gly-Tyr-TrCys)2B] and Au[(TrCys)2B], respectively. The NPs stabilised with the bulkiest PBHs were smaller. This observation may be attributable to the steric bulk of the ligand controlling NP growth. Figure 2 TEM images and size histograms of PBH-capped AuNPs. (a) Au[(Gly-Trp-Met)2B], (b) Au[(Gly-Tyr-TrCys)2B], (c) Au[(Gly-Tyr-Met)2B], (d) Au[(Met)2B] and (e) Au[(TrCys)2B] [Scale bars: 10 nm for (a) and (b); and 5 nm for (c), (d) and (e)].

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