RESUMEN
The present article describes a miniaturized polyacrylamide slab gel electrophoresis-chip (PASGE-Chip) that can rapidly separate a set of predefined samples as well as cell lysate samples for clinical diagnosis. The chip consists of a polymethyl methacrylate (PMMA) upper unit (25 x 30 x 10 mm, width x length x depth) with integrated buffer chambers, running electrodes and loading wells and a bottom unit comprising a silicon dioxide-coated silicon plate with embossed gel chamber (11 x 15 x 0.37 mm). This miniaturized device was designed to be fast, easy to use and cheap to produce. The polyacrylamide slab gel electrophoresis can be performed in less than 10 min with low voltage. The gel-to-gel repeatability is around 3.8%. The limit of detection is approx. 10 ng as determined by Coomassie staining of selected standard proteins, and corresponds to a 10-fold increase in sensitivity as compared with a common size PAGE analysis device (e.g. 10 x 7 cm). The device was successfully applied to peptide mass fingerprint analysis, protein sequencing and ultra-sensitive immunodetection, and the performance was compared to a commonly used regular PAGE device.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/instrumentación , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas/análisis , Secuencia de Aminoácidos , Células Cultivadas , Péptidos/química , Sensibilidad y Especificidad , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We describe a miniaturized instrument capable of performing 2-DE. Our miniaturized device is able to perform IEF and polyacrylamide slab gel electrophoresis (PASGE) in the same unit. It consists of a compartment for a first-dimensional IEF gel, which is connected to a second-dimensional PASGE gel. The focused samples are automatically transferred from the IEF gel to the PASGE gel by electromigration. Our preliminary experiments show that the device is able to focus and separate a mixture of proteins in approximately 1 h, excluding the time required for the staining procedure. On average, the gel-to-gel retardation factor (Rf) variation was 6.2% (+/-0.9%) and pI variation was 2.5% (+/-0.6%). Separated protein spots were excised from stained gels, digested with trypsin, and further identified by MS, thus enabling direct proteomic analysis of the separated proteins.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Lactoglobulinas/análisis , Proteómica , Electroforesis en Gel Bidimensional/instrumentación , Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de Poliacrilamida/instrumentación , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Polímeros/química , Estándares de Referencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
During the European Union project Quantification of Aerosol Nucleation in the European Boundary Layer (QUEST), which began in spring 2003, atmospheric aerosol particles were collected in a Finnish Scots pine forest using a high-volume sampler. The organic compounds in the filter samples were then analysed by on-line coupled supercritical fluid extraction-liquid chromatography-gas chromatography-mass spectrometry (SFE-LC-GC-MS). The sample was first extracted by SFE. During LC the extracts were fractionated into three fractions according to polarity. The final separation was carried out by GC-MS. A fraction volume as high as 840 microL was transferred to the GC, using the partial concurrent eluent evaporation technique. The same instrumentation, with an in-situ SFE derivatisation method, was used to analyse organic acids. Major compounds such as n-alkanes and PAH were analysed quantitatively. Their concentrations were lower than those usually observed in urban areas or in other forest areas in Europe. The wind direction was one of the most important factors affecting changes in the daily concentrations of these compounds. Scots pine needles were analysed with the same system to obtain reference data for identification of biogenic compounds in aerosol particles. Other organic compounds found in this study included hopanes, steranes, n-alkanals, n-alkan-2-ones, oxy-PAH, and alkyl-PAH; some biogenic products, including oxidation products of monoterpenes, were also identified.
Asunto(s)
Aerosoles/química , Cromatografía Liquida/métodos , Cromatografía con Fluido Supercrítico/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Compuestos Orgánicos/análisis , Compuestos Orgánicos/química , Contaminantes Atmosféricos/análisis , Contaminantes Atmosféricos/química , Alcanos/análisis , Alcanos/química , Cromatografía Liquida/instrumentación , Cromatografía con Fluido Supercrítico/instrumentación , Sistemas de Computación , Finlandia , Cromatografía de Gases y Espectrometría de Masas/instrumentación , Pinus/química , Terpenos/análisis , Terpenos/química , Árboles/químicaRESUMEN
Atmospheric particles were collected with a high-volume sampling system at an urban site in Helsinki (Finland). The samples were analysed by on-line coupled supercritical fluid extraction-liquid chromatography-gas chromatography-mass spectrometry (SFE-LC-GC-MS). The aerosol sample was first extracted by SFE. The extract was then transferred to a liquid chromatograph where it was fractionated into four fractions according to polarity. Each fraction from the liquid chromatograph was transferred to a gas chromatograph by large-volume injection, where final separation was carried out. The first LC fraction (280 microl) contained nonpolar compounds, such as n-alkanes, hopanes and steranes. The second fraction (840 microl) included polycyclic aromatic hydrocarbons (PAHs) and alkyl-PAHs, while the third and fourth fractions (840 microl each) contained more polar compounds, such as n-alkan-2-ones, n-alkanals, oxy-PAHs and quinones.
Asunto(s)
Aerosoles/química , Cromatografía Liquida/métodos , Cromatografía con Fluido Supercrítico/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Compuestos Orgánicos/análisis , AtmósferaRESUMEN
Comprehensive two-dimensional gas chromatography utilising a semi-rotating cryogenic modulator was applied to the analysis of urban aerosols. Samples were collected onto glass fibre filters using a high-volume sampler in Helsinki, Finland. Sample preparation included extraction into n-hexane-acetone mixture and clean-up on silver-impregnated silica column. Analyses were performed with GC x GC-FID and GC x GC-QMS equipment. Linearity of the method was good with R2 values greater than 0.994 for all analytes. Polycyclic aromatic hydrocarbons (PAHs) and oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) were identified and quantified in urban particulates. PAH and oxy-PAH concentrations ranged from 0.5 to 5.5 ng/m3.
Asunto(s)
Aerosoles/análisis , Cromatografía de Gases/métodos , Calibración , Espectrometría de Masas/métodos , Salud UrbanaRESUMEN
A laboratory-made cryogenic modulator for comprehensive two-dimensional gas chromatography (GCxGC) was constructed and evaluated. Analytes were trapped with carbon dioxide and desorbed with heated GC oven air. The GCxGC system included a non-polar first-dimension column and a semi-polar second-dimension column connected to a flame ionisation detector. A laboratory-written Matlab-based program was used for the data analysis. Peak widths at half height for n-alkanes, obtained with use of new modulator, were at narrowest 60 ms.