Biomedical subjects

Irene Barinaga-Rementeria Ramírez

Publications and source records attributed to Irene Barinaga-Rementeria Ramírez.

4 recordsLinked to original sources

PubMed2006

Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning.

As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes.

Blotting, Western↗

PubMed2004

Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin-dextran.

A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin-dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin-dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions.

Animals↗

PubMed2004

Affinity two-phase partitioning in acoustically levitated drops.

Miniaturized (<1 microL) biospecific affinity two-phase partitioning in an acoustically levitated drop is described. Miniaturization commonly gives unfavorable surface/volume ratios, but in the levitation approach adsorption problems are minimized since the only surrounding wall is the liquid/air interface of the drop. Biotinylated liposomes were partitioned in aqueous poly(ethylene glycol)/dextran two-phase drops with NeutrAvidin-dextran as the affinity ligand. A two-phase drop was trapped and manipulated in a node of a standing ultrasonic wave. Alternatively, a two-phase system was formed by levitation and evaporation of a polymer one-phase drop. Phase mixing was achieved by adjusting the ultrasonic field and phase separation by readjusting the field. NeutrAvidin-dextran brought about the redistribution of biotinylated liposomes from the poly(ethylene glycol)-rich phase into the dextran-rich phase. Thus, an entire affinity two-phase separation procedure, including mixing of the phases and incubation to allow affinity interactions to develop under constant volume, followed by phase separation under controlled evaporation, can be performed in a single levitated drop. This miniaturized technique would allow the separation of biologically active membranes or organelles from individual cells for analysis.

Journal Article↗

PubMed2002

Affinity partitioning for membrane purification exploiting the biotin-NeutrAvidin interaction. Model study of mixed liposomes and membranes.

Biotinylated negatively charged liposomes as well as membranes were affinity partitioned in an aqueous poly(ethylene glycol)-dextran two-phase system using NeutrAvidin conjugated to dextran as affinity ligand. Both liposomes and membranes redistributed from top to bottom phase upon addition of NeutrAvidin-dextran. The presence of 35-60 mM Li2SO4 was necessary both to force the components into the top phase without ligand and for ligand-dependent redistribution into the bottom phase. Attaching biotin via a hexanamidohexanoyl spacer and an increased density of biotin or NeutrAvidin enhanced the affinity separation. The separation conditions in these model experiments provide a basis for affinity partitioning of membranes using other affinity ligands.

Avidin↗