Surface Density Calculation
Liv Jensen
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol details Surface Density Calculation.
Attachments
Steps
Surface Density Calculation
Quantify the fluorescence of a serial dilution of membrane fluorophore and of proteintag fluorophore in 1% SDS-containing buffer (10micromolar (µM), 6micromolar (µM), 2micromolar (µM), 1micromolar (µM), 0.5micromolar (µM) of each fluor).
Compare the slopes of a plot of fluorescence intensity vs. concentration for the membrane dye and the protein dye to account for differences in optical properties.
Quantify the fluorescence of a series of GUV preparations containing varying mole percentage of membrane fluorophore (0.01%, 0.05%, 0.1%, 0.5%, 1%), using a rectangular selection of an approximately horizontal segment of each GUV.
Calculate the surface density of membrane dye from known surface area of lipids (~0.7nm2 /lipid).
Calculate the slope of a plot of membrane dye surface density vs. fluorescence signal, accounting for intensity from both lipid bilayer leaflets:
Relate surface density of lipid to surface density of protein label by normalizing to signal from bulk solution (from step 2):
