Dear all,
I am working with POPC liposomes and have successfully implemented them in an assay to observe their effect on Beta-Amyloid(1-40) aggregation kinetics.
Now, I am attempting to encapsulate 6-Carboxyfluorescein dye inside POPC liposomes to perform dye leakage assay based on 6-Carboxyfluorescein self quenching properties.
POPC in chloroform dried under vacuum overnight, rehydrated with 10mM TRIS 150mM NaCl buffer, sonicated 30 mins, extrusion (0.1um membrane) 20+ times, DLS confirms good size population ~120nm. I plan to follow this protocol but include 6-Carboxyfluorescein in the hydration buffer, then dialyze away free unencapsulated dye with 'Snakeskin' 10k MWCO dialysis tubing.
MY QUESTION: what is the lowest recommended concentration of 6-Carboxyfluorescein needed to maintain self quenching when encapsulated in liposomes? In literature, I have seen anything varying from 1mM to 50mM.
Thank you!
Dear Clifford,
I have been working with 40mM CF, I think you should not go much lower than that. And keep in mind to match the osmolality of buffer and CF solution.
Good luck
Federica
I'm struggling to actualize the same results. I am using TRIS 10mM NaCl 150nM. I tried to dissolve 20mM CF and it remains supersaturated and insoluble remains. I diluted the sample but insoluble CF still remains all the way down to 1mM CF. I did however perform fluorescence spectroscopy on a serial dilution of the sample, and It shows very strong self quenching all the way down to about 500uM-1mM CF.
My approach was to dissolve the CF in MQ water adding drops of 1 M NaOH just enough to get it solubilised, and checking that pH was about 7.4. Then I used this solution to hydrate the lipid film. To remove the free CF I eluted through a sephadex column using a buffer with matched osmolality to the CF solution.
Thanks for you input, Federica. I actually ran a 20mM CF sample through a 0.22uM filter, and the resulting eluent shows very good quenching. So I will try to use this solution to encapsulate in liposomes.
When I perform Dialysis, I am going to place 1.00mL of extruded liposome solution inside the dialysis bag, and dialyze against 1-2L of buffer (also 10mM TRIS, 150mM NaCl). My question is when I do this, should I expect the volume inside the dialysis bag to increase significantly? Should the bag swell? I am hoping that there is not much volume change, but I should think that the volume will increase due to osmosis from the CF attempting to equalize concentration? or will the un-encapsulated CF escape freely without change of volume inside dialysis bag?
Unfortunately I have no experience with dialysis. I just found a good explanation in this paper of a protocol similar to the one I used:
DOI: 10.1038/NNANO.2012.84
The concentration that I used is 50mM. When you dissolved the fluorescein, you should use NaOH to adjust the pH to ~7.4,so the solution may turn to clear dark orange. And same to Federica, the liposomes were purified in the Sephadex50 column.
Here is the article that clarify the 6CF concentration that affect the fluorescence.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts