Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 12923764

Prefractionation techniques in proteome analysis.

Pier Giorgio Righetti·Annalisa Castagna·Ben Herbert·Frederic Reymond·Joël S Rossier
View original source ↗Export metadata ↓

Abstract

The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis. In the first case, Fountoulaki's groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins. As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps. The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

Explore related subjects

Chemical FractionationChromatography, LiquidElectrophoresis, Gel, Two-DimensionalProteomeJournal ArticleResearch Support, Non-U.S. Gov'tReview

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Pier Giorgio Righetti, Annalisa Castagna, Ben Herbert, Frederic Reymond, Joël S Rossier. 2003. Prefractionation techniques in proteome analysis.. https://doi.org/10.1002/pmic.200300472

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Improved procedure for separation and purification of Arthronema africanum phycobiliproteins.

A rapid, inexpensive and reliable procedure for separation and purification of C-phycocyanin (C-PC) and allophycocyanin (APC) from Arthronema africanum based on a previously described rivanol-sulfate method for C-PC purification was developed. Exclusion of NaCl from the extraction buffer resulted in complete separation of APC and C-PC, two-fold reduction of rivanol treatments, and a higher yield and purity of C-PC. Pure C-PC (A(620)/A(280) of 4.52) and APC (A(652)/A(280) of 2.41) were obtained. The estimated molecular masses of the alpha and beta subunits were 17 and 19 kDsmall a, Cyrillic for capital ES, Cyrillic-phycocyanin and 16 and 18 kDsmall a, Cyrillic for APC, respectively. The overall C-PC recovery of 55% (w/w) from its content (100 mg) in the crude extract was 10-20% higher than so far reported. The procedure appears promising for scaling up and broader applications.

Chemical Fractionation↗
PubMed2007

Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction.

In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.

Chemical Fractionation↗
PubMed2007

Effect of solvent concentration on the extraction kinetics and diffusivity of Cyclosporin A in the fungus Tolypocladium inflatum.

The kinetics of solid-liquid extraction and extraction yields of the immunosuppressant drug Cyclosporin A (CyA) from the mycelia of Tolypocladium inflatum were examined in this study. A 2 L stirred, baffled vessel was used to extract CyA from wet mycelia mass. Three different organic solvents were used, namely, methanol, acetone, and isopropanol at different concentrations in aqueous mixtures at room temperature. It was found that the best solvent was acetone at 50% v/v concentration achieving 100% extraction of CyA from the mycelia of T. inflatum. Although acetone proved to be the better solvent for CyA extraction, further studies were performed using methanol. A linear relationship was found between extraction yield of CyA and methanol concentration with 100% CyA extraction at 90% v/v methanol. The partition coefficients of CyA between the solid mycelia phase and the aqueous solvent phase were found to decrease exponentially with increasing methanol concentration. A liquid extraction model was developed based on the diffusion equation to correlate the kinetic data of CyA extraction from the solid mycelia of T. inflatum. Non-linear regression analysis of experimental data was used with the diffusion equation in order to calculate the effective diffusivities of CyA in the mycelia of T. inflatum. For all three organic solvents used, the effective diffusivities of CyA were found to be between 4.41 x 10(-15) and 6.18 x 10(-14) m(2)/s. This is the first time CyA effective diffusivities in T. inflatum are reported in the literature.

Chemical Fractionation↗