I use FluoZin-3 fluorescent dye to test the transport activity of a membrane transport protein. I need help to separate proteoliposome from the excess fluorescent dye which remain outside. My proteoliposome consist of E.coli polar lipids and egg yolk phosphatadylecholine in 3:1 (w/w) ratio and protein:Lipd ratio is 1:25. After making the proteoliposome where FluoZin-3 (20uM) is trapped inside the proteoliposome by ultrasonication, I use ultracentrifugation (160000xg) to pellet down proteoliposome. However, after centrifuging, in the middle of the pellet, I get a very intense dot which is having the color of the dye. This can only be seen in the proteoliposome but not in the control where I don’t have my protein. I wonder whether, my proteoliposome is burst during the ultracentrifugation or my protein is messed and precipitated by the dye. Is anybody having any idea what is happening and any suggestion to prevent this problem? (Both proteoliposome and the control are in a MOPS buffer pH = 7.0, 100mM NaCl, 1mM DTT and 20uM FluoZin-3).
Thank you.
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