Biology Reference
In-Depth Information
A
B
Extruded liposomes
1
1
Saturated with detergent
2
2
Dilution below cmc
Purified,
solubilised
receptors
added
3
Biobeads
removed, proteo-
liposomes formed
Dialysis
Biobeads added
3
Proteoliposomes
formed
FIGURE 18.1
Reconstitution by dilution and dialysis (A) and by using Bio-beads for detergent removal (B).
(A): (1) Lipids, detergent, and protein are mixed at the desired ratios and incubated for 1 h.
(3) The reconstitution mixture is rapidly diluted below the cmc of the detergent. (4) The
detergent monomers are removed by extensive dialysis. (B): (1) Resuspended liposomes are
destabilized with detergent. (2) Detergent-solubilized protein is added to the required
concentration and the mixture incubated for 1 h. (3) Detergent is removed with Bio-beads at a
10:1 Bio-bead-to-detergent ratio (w/w).
ratios (
Rigaud et al., 1997
), and also because of the relative rapidity of the method
compared to dialysis, resulting in a larger proportion of functionally reconstituted re-
ceptors. However, lipid adsorption to the beads can be up to 30%, so if a low protein-
lipid ratio is important, this possibility needs to be accounted for or the dialysis method
used. However, size homogeneity of proteoliposomes formed using Bio-beads is far
greater than for the dialysis method).
18.1.4.1
Protocol for reconstitution using Bio-beads
Reagents:
1.
Stocks of extruded liposomes at 5 mg/ml in liposome buffer (50 mM Tris pH 7.4,
100 mM NaCl, 1 mM EDTA)
2.
Purified protein stocks in detergent (0.1% DDM, 0.01% CHS)
