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Der p 1 is the main allergen of house dust mite Dermatophagoides pteronyssinus, which has routinely been detected in residential dust. However, the procedure for extracting Der p 1 from reservoir dust has not been well defi ned. The aim of this study was to compare Der p 1 mass fractions in dust extracts prepared using the following extraction buffers: phosphate (pH 7.4), borate (pH 8.0), and ammonium bicarbonate (pH 8.0), all with 0.05 % Tween 20. Twenty-eight dust samples were divided into three aliquots and each portion was extracted with one of the three buffers at room temperature. Der p 1 mass fractions were measured in a total of 84 dust extracts using the enzyme immunoassay (range: 0.1 μg g-1 to 7.53 μg g-1). Statistical methods including intraclass correlation showed a high agreement between Der p 1 mass fractions irrespective of the extracting medium. Our results suggest that all three buffers are suitable for the extraction of mite allergens and routine Der p 1 analysis in dust.

Genomic DNA extraction Second-stage juveniles (J 2 ) were concentrated by centrifugation at 3 000 rpm for 5 min, and the supernatant was discarded. The precipitated pellet in Eppendorf tubes was frozen with liquid N 2 and macerated using a micropestle. The macerate was homogenized with 250 μL of extraction buffer [(500 mM KCl, 100 mM Tris HCl pH 8.0, 1 % Tritón X-100, 400 μg/mL Proteinase K (Sigma-Aldrich ® )], and then this mixture was incubated at 60 °C for 4 h and the end at 90 °C for 10 min to inactivate the Proteinase K. Subsequently, the mixture was centrifuged

diagnostics on dry blood spots (DBS) for neuronal ceroid lipofuscinoses, by simultaneous fluorimetric and MS-MRM analysis (Figure 1). Figure 1. Schematic representation of MRM-MS diagnostics of NCLs Acknowledgements: This work was supported by the strategic grant POSDRU/159/1.5/S/137750 * Corresponding author, e-mail address: ion.laura26@yahoo.com 3 mm Ø Protein saver card Dry Blood Spot (DBS) 85 µL extraction buffer Incubation for 45 min at 37o C 75 µL substrate Incubation for 24 h at 37o C 50 µL I.S. in FAMRM – MS measurement m/z P IS

for 45 min at 4ºC as previously described ( 23 ). The proportion of the dead cells (stained ones) were quantified under bright light with an optic microscope Leitz Laborlux 12. The average of percentage of dead cells was obtained by three independent experiments with two technical replicates. A minimum of 2,000 cells were counted per replicate. Caspase 3 activity assay Total proteins were extracted from in vitro samples grounded in extraction buffer (50 mM HEPES pH 7.4, 5 mM CHAPS, 5 mM DTT), and protein concentration was determined by Bradford method, using the

. For each sample, 0.3–0.5 g were weighed in microtubes and thawed in 800 µl extraction buffer [50 mM Tris, 2mM DTT, 1 mM EDTA, 1 g. l –1 Triton X 100, pH 7.5]. The mixtures were homogenized and centrifuged for 10 min at 10.000× g . The extractable soluble protein content of the resulting supernatant was determined based on the method of Bradford (1976) using the Bio-Rad protein assay dye reagent concentrate (catalog number: 500-0006). The results were used to calculate the protein degradation rate of the samples and the supernatant was further used for gel

-lyase The enzyme extracted from 300.0 g leaf tissues using 6.5 ml of 50.0 mM Tris-HCl buffer (pH 8.8) containing 15.0 mM of ß-mercaptoethanol ( 27 ). The resulting homogenate centrifuged at 10000.0 g for 30.0 min. The amount of protein in the supernatant assayed by the method of Lowry et al. 1951( 25 ). The activity of the enzyme calculated on the basis of formation of cinnamic acid. The reaction mixture contained 1.0 ml extraction buffer, 0.5 ml of 10.0 mM L-phenylalanine, 0.4 ml of Milli Q water and 0.1 ml of enzyme extract. The reaction carried out at 37 °C for 1h and

were performed in triplicate. Preparation of crude extract from plant samples The plant samples were homogenized in an extraction buffer which consisted of 50 mM sodium phosphate (pH 7.0) buffer with 0.1 mM ethylenediaminetetraacetic acid (EDTA). The homogenates were filtered and then centrifuged at 14,000 g for 15 min at 4 °C. The process continued by collection of supernatants, which were then used as a crude extract for the assays. To minimize a possible activity loss risk, extract preparation and enzyme activity assays were performed on the same day