I have been working on supported lipid bilayers on glass slides for sometime now. I use pure POPC (neutral lipid) for my experiments. The issue is that the diffusion coefficients i get is less than what is reported in literature for the fluorescent probe i am using. I get values of D around 1 um2/sec whereas literature reports values like 2.5. I use 50 mM HEPES Buffer,0.1 mM EDTA, pH 7.4. The bilayers look fine and coverage in glass slide is 100%. I bleach the probe (0.5 mol%NBD-PC) upto background in 1.5 seconds and monitor recovery for 8 minutes. I use Soumpasis model for fitting...Do any of you use a similar system and get the same values? I cannot go shorter than 1.5 seconds for bleach time.
Agreed with Pedro. Exact values are difficult to reproduce with SLB experiments because of difficult-to-control influences from the substrate. Its possible that a polymer cushion would get you to the literature reported value, but I think you're in the right range anyway. It is more important that you are getting full recovery, because this means you have a truly fluid bilayer without many defects.
Best of luck.
We looked at diffusion in such bilayers by single molecule tracking of fluorescent lipid analogs. Preferentially in pure POPC. We often see binding events of the probes, which certainly should not be there for really perfect, fluid bilayers. As Ilya said, there are always imperfections and an influence of the substrate. PEG polymer cushions do improve the situation a bit, but not perfectly.
First question: are you sure that YOUR data are wrong ? You may find in literature different results but looking carfully to them you will see that measurements were not always made rigorously.
If you are sure that something is wrong with your data, there are several possible reasons:
a) What about your POPC powder or solution? If air is present, if the vial is open for some time, not under N2, you can have some reticulation of the lipid by reaction of the double bound= slow down of the diffusion.
b) It can be the cleaning of the support. What do you use, Piranha, detergent,..? Forget it! Use fresh alcolic NaOH during 10'under gente stirring, wash extensively with ultrapure pure water and your support will be hydrophiic, D will be reproducible from one experiment to another and should give you the right value.
To test one possibility versus the other, you can carry an experiment with DLPC (no double bound and fluid at all temperaures). Doing that you check independantly one parameter.
I also fought hard against this issue, more precisely it was non reproductibility. The lipid was DMPC. Since I understood and built up this cleaning method, measurements are perfectly reproducible. This summer I worked with POPC (which has a double bound) and we have great problems and discover to flexible handling;
Working on supported bilayer belongs to Nanosciences ( 1 nm between the support and the bilayer). In you do not work very very cleanly and methodically, you waiste your time.
Good luck,
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