Generation of proteoliposomes from subcellular fractions

Abstract

Intracellular membranes are highly dynamic, yet they retain their identity and functional characteristics. Integral membrane proteins, which must confer this specific membrane identity, remain poorly characterized at the biochemical level, largely because detergent-mediated solubilization is required for purification and analysis, and several properties of integral membrane proteins can only be investigated when the molecule is properly embedded in a lipid bilayer. We present a method for the efficient reconstitution into proteoliposomes of integral membrane proteins from subcellular fractions. Integral membrane proteins were identified on high-resolution two-dimensional gels after selective extraction of soluble and peripheral membrane proteins; they accounted for 8% of the number of resolved polypeptides. A reconstitution procedure based on membrane solubilization with dodecyl-octaoxyethylene (C12E8) and subsequent detergent removal with BioBeads SM-2 resulted in the efficient reconstitution of several membrane proteins into proteoliposomes of uniform density. The generated proteoliposomes strongly resemble the starting membrane fraction in protein composition. This reconstitution allows the functional characterization of integral membrane proteins after enrichment and/or specific (immuno)depletion.

Surfactant-Mediated Cloud Point Extractions: An Environmentally Benign Alternative Separation Approach

“…Electrochemical detection is an ideal alternative since the typical surfactants employed in the CPE technique are relatively electroinactive. ,,, In many applications, the surfactant can be removed from the surfactant-rich phase via a variety of techniques. For surfactants with a relatively high cmc (>1 mM), the surfactant can be efficiently removed by dilution and dialysis ,, (usually within ≈24 h; thus, for instance, ca. 90% of Trition X-114 was removed after 12 h by dialysis) .…”

“…90% of Trition X-114 was removed after 12 h by dialysis) . For surfactants with low cmc values, contact with synthetic beads such as Bio-Beads SM2, a copolymeric material with an affinity for Triton-type surfactants, , or Amberlite XAD-2 can be utilized to remove the surfactant . The detergent can also be removed by passing the surfactant-rich phase through a column of materials such as Bio-Gel HTP, hydroxyapatite, silica gel, or Florisil, , as well as combinations of these.…”

“…It is anticipated that a comprehensive validation of the CPE method for these vitamins will be developed. Numerous other examples (Table ) using Triton X-114 (or other surfactants) and the CPE technique have been reported in the recent literature. ,,− ,,,, …”

Extração Por Ponto Nuvem: Técnica Alternativa Para a Extração De Resíduos De Agrotóxicos

“…A maioria dos surfactantes não-iônicos é, provavelmente, melhor extraída com solventes mais polares ou pela mistura de solventes polares. O método mais comum e mais fácil de remover o surfactante é a diálise (ROJO et al, 1997). Esse método, simples e eficaz para surfactantes com valor de cmc relativamente alto (acima de 1 mmol L -1 ), é muito empregado para a remoção da maioria dos surfactantes zwitteriônicos.…”

The transmembrane protein p23 contributes to the organization of the Golgi apparatus

“…Detergent concentration was then adjusted to 3% (w/v) by addition from a 20% stock in water. Membranes were solubilized by repeated pipetting for 5 minutes at room temperature, and insoluble material was removed by centrifugation (4 minutes, 30 psi ≈135000 g, k=11) in a Beckman Airfuge (Rojo and Wallimann, 1994;Rojo et al, 1997a).…”