I was wondering how many of you work with membrane proteins and might me able to help out with this one.
I'm trying to reconstitute mitochondrial carriers into liposomes. The established protocol is done in 3 major steps:
a) isolating the protein, of course, from bacterial strain after expression. the isolation is basically from inclusion bodies and solubilization in 1% sarkosyl in tris buffer.
b) making the proteoliposomes, which is done incorporating the protein in extruded egg yolk phospholipids (9mg/mL), internal substrate (ADP or ATP in my case, 30mM), Tx-114 and cardiolipin in 10mM buffer MOPS, 50mM NaCl. this mix is passed through a Amberlite XAD-2 column, 24X
the un-incorporated internal substrate is eliminated using another column, Dowex AG1-X8, equilibrated with the same buffer MOPS as before
c) the transport assay is done mixing the liposomes with ATP (3H) and stoping the reaction with 30mM pyridoxal 5’ phosphate and 10mM bathophenanthroline after 0,2,5 & 10minutes.
excess of non-trasported radiactivity is eliminated using sephadex G-75 equilibrated with the same Mops buffer as before.
So far, no transport has been observed. I was wondering if any of you could shed some light on the subject.