A quantitative western blot technique using TMB: Comparison with the conventional technique

Numerous molecular biological experiments performed throughout the world require the detection or quantification of a protein of interest. Western blotting is one of the most popular techniques used for this purpose and offers quantitative information with the aid of specialized software. However, its dependence on the picture that is captured, and the background and the absence of a common protocol prevent the technique from being completely quantitative. To overcome these obstacles, we present a simple and reliable assay that is similar to the regular technique, with the exception of the last stage of band visualization and quantification. We propose that small pieces of the blot that include the protein of interest can be cut and dipped in a small volume of 3,3',5,5'-tetramethylbenzidine solution, giving a colorimetric signal with linear dependence on the quantity of the protein. The reaction is stopped with H₂SO₄, and the signal is measured in a plate reader. This modification shows high linearity without additional costs and can be applied for both purified proteins and proteins found in a lysate. The results obtained with our proposed technique were compared with those obtained by the conventional method and proved to be more reliable.     

Antioxidant and chemical properties of Inula helenium root extracts

The objective of the paper was to investigate the chemical composition of Inula helenium roots extracts and to evaluate the antioxidant potential conferred by the chemical constituents. GC/MS and HPLC/MS techniques were used to characterize two extracts separated from Inula helenium roots by extraction with chloroform and ethyl acetate, respectively. Volatile compounds have been identified by GC from their mass spectra and retention time values, while HPLC identification of phenolic compounds was realized by comparing their retention times, UV and MS spectra with those of standards or literature data. Measurements of antioxidant activity of Inula helenium root extracts showed a variation between them, which can be correlated with the flavonoid and total phenolic contents. Both Inula helenium root extracts contain phenolic acids (caffeic, chlorogenic, dicaffeoyl quinic, hydroxibenzoic), terpenes (alantolactone) and different flavonoids (epicatechin, catechin gallate, ferulic acid-4-O-glucoside, dihydroquercetin pentosyl rutinoside, kaempherol-7-O-dipentoside, quercetin-3-O-β-glucopyranoside). In addition, the study provides preliminary data on the anti-inflammatory activity of Inula helenium root extracts, this being evaluated using the fresh egg albumin as phlogistic agent, and aspirin as reference compound. Root extracts of I. helenium did not exert any significant anti-inflammatory effect on egg albumin-induced rat paw edema.      

L'influence de la temperature sur l'echange anionique

The influence of temperature on the distribution coefficients of Zn ions in systems ethanol or acetone, hydrochloric acid and Dowex 1×8 ion exchange resin (100–200 mesh) was studied. TheK _d-values increase a little with the temperature. The thermodynamic constants for some systems were calculated.      

Enzymatic degradation of some nanocomposites of poly(vinyl alcohol) with starch

A study of the enzymatic degradation of some montmorillonite-containing nanocomposites of poly(vinyl alcohol) with starch was based on the determinations of mass loss and the reducing sugars. The degraded residues have been examined by FT-IR spectroscopy and optical microscopy. It has been established that the nanoparticles hinder degradation, while the susceptibility to enzymatic degradation varies in the order: PVA/starch/nanocore > PVA/starch/Bentonite > PVA/starch/Peruvian clay.     

Development and validation of a cellular biosensor detecting pesticide residues in tomatoes

Two of the most important categories of pesticides used in agricultural practice are organophosphates and dithiocarbamates. Their extensive and inappropriate use has rendered their reliable monitoring at trace levels more and more necessary. This study presents the construction of a rapid and sensitive cellular biosensor test based on the measurement of changes of the cell membrane potential of immobilized cells, according to the working principle of the Bioelectric Recognition Assay (BERA). The cells were immobilized by entrapment in a sodium alginate bead and directly applied in different pesticide dilutions and agricultural samples. The pesticides used were the organophosphate insecticide diazinon and the dithiocarbamate fungicide propineb. Two different cell types, N2a (neuroblastoma) and Vero (fibroblast) were used as the biosensory elements in order to investigate their differential response against the pesticides. In this way, we hoped to increase the selectivity of the assay. Based on the observed patterns of response, we demonstrate that the sensor can be used for the qualitative and, in some concentrations, quantitative detection of the pesticides with a high degree of reproducibility. The lowest detected concentration was 3 nM. Finally, for the investigation of the effects of different pesticides on the accumulation of cytosolic Ca²⁺, we conducted a fluorescent assay on N2a cells treated with tomato sample extracts, which were replicates of the E.U. proficiency test sample. The tomato samples were either organically grown or contained 14 different pesticides. The experimental results showed a higher increase of the intracellular Ca²⁺ concentration in cells treated with non-organic samples compared to the cells treated with organic samples.     

Thermal degradation of keratin waste

Thermal degradation of sheep wool, human hair and chicken feathers was studied by TG-MSD/FTIR and by pyrolysis followed by GC-MSD analysis in order to identify the degradation compounds and the temperature range in which they are formed. Only small differences were found between the studied keratin samples. They consist mainly in shift of characteristic temperatures of degradation and in relative amounts of compounds in degradation products, especially in aqueous phase. Degradation started with formation of ammonia and CO₂ (from 167 and 197 °C respectively and with maximum evolution at 273 and 287 °C respectively), continues with formation of sulphur-containing inorganic compounds (SCS, SCO, H₂S and SO₂ at 240, 248, 255 and 253-260 °C respectively) and of water (255 °C). Thiols are formed in two stages (257 and 320 °C) while the evolution of nitriles is maximum around 340 °C and continues up to about 480 °C. Phenol and 4-methylphenol are the most important degradation compounds, formed at 370 and 400 °C respectively. Nitrogen was present mainly in aliphatic/aromatic nitriles, pyrroles, pyridines and amides while sulphur was found mainly as sulphides, thiols, thiazoles and thiophenes.     

A thermogravimetric study of structural changes of lime wood (Tilia cordata Mill.) induced by exposure to simulated accelerated UV/Vis-light

A series of experiments were carried out to investigate photodegradation of lime wood (Tilia cordata Mill.) during artificial UV/Vis light irradiation for 600 h. Photodegradation of the wood samples was evaluated by thermogravimetry throughout the irradiation period at an interval of 100 h.The alteration of wood was observed by means of the peak temperatures of DTG variation and by the mass losses observed during heating, evaluated on the basis of the measured thermal data. The results obtained indicate a shifting of the DTG maxima to lower temperatures which may be related to the decreasing in the stability components during photodegradation. The values of the overall activation energies corresponding of the decomposition process progressively decreases.The decreased temperatures decomposition processes, weight losses and integral area of the peaks assigned to lignin, the increased integral area of the peaks assigned to hemicelluloses could be explained by new formed structures, mainly due to formation of reactive species.     

Improved confidence intervals for quantiles

We derive the Edgeworth expansion for the studentized version of the kernel quantile estimator. Inverting the expansion allows us to get very accurate confidence intervals for the pth quantile under general conditions. The results are applicable in practice to improve inference for quantiles when sample sizes are moderate.