Gel‐free IEF in a membrane‐sealed multicompartment cell for proteome prefractionation

Abstract

A minidevice for performing gel‐free proteome prefractionation via conventional IEF in soluble carrier ampholyte buffers is reported here. It consists of a compact block of polyoxymethylene in which eight samples and two electrode chambers are machined. Each of the eight sample chambers can be filled with up to 120 μL of sample and has the following size: 7 mm width, 3 mm depth and 10 mm height. The anodic and cathodic compartments have the same width and height as the sample chambers, but with a depth of 6 mm, thus accepting up to 250 μL of electrodic solutions. Focusing is in general accomplished in 2 h with a voltage gradient of up to 1000 V (7 cm electrode distance). Easy fractionation and collection of the content of the eight chambers is achieved by simply pressing a rubber diaphragm against the edges of the thin walls separating each well, this automatically breaking liquid continuity. The performance of this device has been tested by subfractionating total cell lysates of a human cancer cell line (U2Os) and of Escherichia coli bacterial cells, and by analysing the content of each chamber by mono‐dimensional SDS‐PAGE and 2‐D maps.

Bacterial adaptation to life in association with plants – A proteomic perspective from culture to in situ conditions

“…Similar approaches based on protein pre‐fractionation either gel‐free (Off‐gel) 24 or with ion‐pair RP chromatography 25 are emerging techniques and will be of sure useful in the near future for the investigation of bacterial/plant interactions.…”

Bacterial Electron Transfer Chains Primed by Proteomics

“…After SDS-PAGE, the gel is cut into multiple pieces, proteins in the pieces are digested in-gel and digests are subjected to LC-MS/MS separately. This preseparation may also involve offline IEF, common protein separation methods such as anion exchange or affinity column chromatography, CE as well as methods that have been specifically designed for tandem MS proteomic analyses (Cologna, Russell, Lim, Vigh, & Russell, 2010;Freeman & Ivanov, 2011;Gilmore & Washburn, 2010;Lam, Antonioli, Righetti, Citterio, & Girault, 2007). Obviously, this method can be applied to lysates of whole cells as well as of subcellular fractions, which have been enriched or fully purified by designated procedures.…”

Protein and peptide fractionation, enrichment and depletion: Tools for the complex proteome

“…Lam et al have recently described an even lower volume (120 mL/fraction), multi-celled device that uses CA-generated gradients similar to the Rotofor [153]. Here, proteins are focused in solution, which is allowed to flow laterally over Eluting multiply phosphorylated species can be difficult Non-specific binding of acidic species overcome by methyl esterification of peptides [104] Heparin affinity chromatography Heparin (linear polymeric sulphated glycosaminoglycan) [33] Broad range of proteins including (anti)coagulation factors [105], protein synthesis and growth factors [33], enzymes [106,107] Breast cancer MCF-7 cells [108], serum [109] Proteins bind to heparin based on biological interaction/specificity [106] High salt elution is incompatible with 2-DE.…”