The name is absent

binding onto the lipid is also insufficient to account for the short range deviation. As a
control, we will show that for silicon nitride, which contains immobile surface charges,
short range deviations can be accounted for using an AFM tip-induced counterion
binding charge regulation model. Finally, we will show that a mobile lipid charge
regulation model explains the behavior of the AFM data over lipids, in the short-range.
Furthermore, force curves over immobile gel-phase lipids are shown not to exhibit the
short-range deviations seen in highly mobile fluid-phase lipids.

3.2 Experimental method

3.2.1 Preparation of DOPC:DOPS supported lipid membranes

Following the general methods described in [1], Lyophilized dioleoylphosphatidylserine
(DOPS-anionic lipid) and dioleoylphosphatidylcholine (DOPC-Zwitterionic lipid)
obtained from Avanti Polar Lipids, Alabaster, AL, were dissolved in chloroform and
mixed at various relative mole fractions. The mixtures were dried under nitrogen gas,
placed under low vacuum for at least 1 h, and then hydrated with deoxygenated double
deionized water for a final lipid concentration of ~2 mg∕mL. The lipid solutions stood
overnight in a dark, room-temperature environment followed by vigorous agitation for at
least 1 h. The resulting multilamellar vesicle solutions were refrigerated and stored for up
to two weeks. Supported lipid bilayer membranes for AFM analysis were formed on mica
substrates by vesicle fusion [17]. A IOO mL drop of the multilamellar vesicle solution at a
lipid concentration of 20-200 mg∕mL (diluted from stock in double deionized water) was
placed on the mica for 20 min at 35-40 ⁰C. The sample was then rinsed with double

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