Hi,
I am a postdoc working with proteoliposome. However, I have not been able to make proteoliposome with my membrane protein. Here is my general procedure.
a, dry the lipid as the thin film on the bottom of the tube
b, add buffer to make lipid suspension
c, go through 200 um filter in a extruder
d, add detergent (DM, triton x100)
e, add protein
f, use dilution method to remove the detergent, spin down and resuspend the pllet. Or, use biobeads 3 times to remove the detergent
g, add external pyranine and test the proton pumping
I have some questions as follows.
1, the choice of lipids. Is the transition temperature important? If I use DOPC, since it has low transition temperature, after I add detergent (DM) and protein, and use two methods to remove the detergent, the liposome is still leaky, and therefore there will not be any pumping activity. Hence, do I need to use a lipid with greater transition temperature, so that after detergent removal, the liposome is sealed?
2, when I add detergent and also remove detergent, do I need to work in a temperature higher than transition temperature?
Thanks
You could try to use DMPC which has a melting temperature about 23 degrees. and do the whole reconstitution in the cold room instead. Doing reconstitution with gel phase lipids makes it easier for the liposomes to form. Perhaps you could try DDM or NMG3 as surfactant, specially NMG3 is often quite good for membrane proteins. Check the following paper:
Nature Methods 7, 1003–1008 (2010).
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