Affinity partitioning for membrane purification exploiting the biotin–NeutrAvidin interaction

Ramírez, Irene Barinaga-Rementeria; Mebrahtu, Sofia; Jergil, Bengt

doi:10.1016/s0021-9673(02)00841-5

Cited by 8 publications

(7 citation statements)

References 19 publications

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“…Liposomes have been used as a system to evaluate the effect of different parameters on the affinity partitioning of membranes. [23][24][25] This investigation illustrates that miniaturized two-phase systems can form and be manipulated in levitated drops and that the distribution of biotinylated liposomes is determined by the presence of NeutrAvidin-dextran.…”

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confidence: 74%

“…Small unilamellar PC vesicles containing 2% (w/w) of biotinylated lipid (biotin-LC-LC-DPPE) were prepared by sonication . Biotin-LC-LC-DPPE was synthesized by coupling synthetic DPPE to EZ-Link NHS-LC-LC-biotin …”

Section: Methodsmentioning

confidence: 99%

“…To explore the potential of levitated drops for biospecific affinity partitioning in aqueous two-phase systems, biotinylated liposomes were partitioned in aqueous poly(ethylene glycol)/dextran two-phase drops with NeutrAvidin (a modified form of avidin that also binds biotin) conjugated to dextran as affinity ligand. Liposomes have been used as a system to evaluate the effect of different parameters on the affinity partitioning of membranes. − This investigation illustrates that miniaturized two-phase systems can form and be manipulated in levitated drops and that the distribution of biotinylated liposomes is determined by the presence of NeutrAvidin−dextran.…”

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confidence: 97%

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Affinity Two-Phase Partitioning in Acoustically Levitated Drops

et al. 2003

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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.

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confidence: 74%

Section: Methodsmentioning

confidence: 99%

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confidence: 97%

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Affinity Two-Phase Partitioning in Acoustically Levitated Drops

et al. 2003

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“…Furthermore, an analogous immunoaYnity approach has been applied successfully to adsorb cells and membranes to avidinylated solid phase [25], indicating that the biotinavidin interaction is suYcient for binding in this approach. This is important as we have found in model aYnity partitioning experiments that the binding between biotin exposed on a negatively charged liposome surface and NeutrAvidin-dextran may be restricted by electrostatic repulsion [15,16]. It is clear that the biotin moiety coupled to the secondary antibody is accessible for strong interactions with NeutrAvidin-dextran.…”

Section: Discussionmentioning

confidence: 98%

“…Caveolae are specialized membrane structures present as plasma membrane invaginations in many cell types [12], most likely involved in endocytosis, transcytosis, and signal transduction [13,14]. The approach was based on conditions for aYnity two-phase partitioning utilizing the biotin-NeutrAvidin interaction [15][16][17]. To make the aYnity method readily applicable to various antibodies an immunoaYnity sandwich technique was explored; the speciWc primary antibody was allowed to bind a biotinylated secondary antibody, which, in turn, interacted with NeutrAvidin-dextran in the two-phase system.…”

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confidence: 99%