reconstitution of membrane proteins into liposomes - (Nov/03/2009 )
i've been working with a mitochondrial carrier with aims to reconstitute it into liposomes. After the protein has been expressed in bacteria, I solubilize it with ~3% TX-100. The recontitution takes place mixing protein, PC, cardiolipin, TX-114 & internal susbtrate to further load the mixture (x24) into an Amberlite column (anion exchanger).
Excess of external substrate is removed using a Sephadex G75. The transport assay is performed after this.
So far I have been unable to observe transport using this conditions. Does anyone have any experience/comments/advice for my issue
Reconstitution into liposomes can be tricky. Sounds as if you're trying to remove the excess detergent on the column.
Do you have any indication that there are closed membrane bodies? Proteoliposomes are usually turbid. If the solution is not opalescent to the eye, take a laser pointer and check if you can see the light beam - scattered light (poor man's static light scatter instrument). If there's no scatter there are no proteoliposomes; you may still be dealing with detergent micelles. Another quick and dirty test: does the solution readily produce bubbles. You can test this by pipetting a few microliter of air into the solution and compare the appearance and behavior of the bubbles before/after the column reconstitution. If you still have a lot of detergent around you'll see plenty of foamy bubbles.
My 2 ct,
Protein solubilization tools